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Carcinogenic Potential of Benzene and Toluene When Evaluated

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  • Carcinogenic Potential of Benzene and TolueneWhen Evaluated Using Cyclin-dependentKinase Activation and p53-DNA BindingCraig Dees,' Minoo Askari,2 and Don Henley2'Risk Analysis Section, Health Sciences Research Division, Oak RidgeNational Laboratory, Oak Ridge, Tennessee; 2University of Tennessee,Knoxville, Tennessee

    Benzene is carcinogenic, whereas toluene is thought to have little carcinogenic potential.Benzene and toluene were found to activate cyclin-dependent kinase 2 in rat liver epithelial (RLE)and HL60 cells. pRbl 05 was hyperphosphorylated in RLE cells treated with either solvent. Kinaseactivation and subsequent hyperphosphorylation of pRbl 05 and p53 by benzene or toluene maybe responsible for their growth promotional effects, but it does not account for increasedpotential of benzene to induce cancer. Therefore, we examined the ability of these solvents toincrease p53-DNA site-specific binding in RLE cells. Benzene increased p53-DNA site-specificDNA binding in RLE cells compared to control levels or the effects of toluene. Increasedp53-DNA site-specific binding by benzene may be caused by damage to cellular DNA. If so,although both solvents appear to have promotional activity, the increased potential of benzene todamage DNA may be responsible to the difference in the ability of benzene to cause cancer.

    Environ Health Perspect 104(Suppl 6)11289-1292 (1996)

    Key words: p53, pRbl 05, tumor promoter, phosphorylation, DNA damage, cyclin-dependentkinase

    IntroductionRecently, we showed that benzene, toluene,and chloroform induce hyperphosphoryla-tion of the tumor-suppressor gene productp53 (1). Hyperphosphorylation of p53 andpRb105 by promoters like 12-O-tetrade-canoylphorbol-13-acetate (TPA), benzene,toluene and chloroform is probably due totheir effects via protein kinase C (PKC) orby inducing a cascade effect in kinasesresponsible for signal transduction (1-4).Stimulation of kinase activity and the sub-sequent hyperphosphorylation of tumor-suppressor gene products, may partiallyaccount for the promotional effects exhib-ited by benzene, toluene, and chloroform.Promoter attenuation of p53's ability toinduce cell cycle arrest may also keep p53from preventing the replication of damaged

    DNA (5). Therefore, posttranslationalmodification of p53 or other tumor-sup-pressor gene products induced by benzeneor toluene may also be a factor in deter-mining the relative carcinogenicity of thetwo solvents.

    Since benzene and toluene both appearto have adverse effects on molecular mecha-nisms that control cell growth and preventthe duplication of genetic errors, theseeffects do not explain the apparent differ-ences in the ability of the two solvents tocause cancer. It is clear that benzene is a car-cinogen, but toluene is thought to be eithernoncarcinogenic or to have little potentialto induce cancer in hematopoietic cells.

    To resolve the difference in the carcino-genic potential of benzene and toluene, we

    This paper was presented at Benzene '95: An International Conference on the Toxicity, Carcinogenesis, andEpidemiology of Benzene held 17-20 June 1995 in Piscataway, New Jersey. Manuscript received 16 January1996; manuscript accepted 14 June 1996.

    Oak Ridge National Laboratory is managed by Martin Marietta Energy Systems, Inc., under contract DE-AC05-840R21400 with the U.S. Department of Energy. This paper has been authored by a contractor of theU.S. government under contract DE-AC05-840R21400. This work was supported by Laboratory DirectedResearch and Development Funds, Department of Energy. We appreciate materials supplied by J.E. Trosko ofMichigan State University and Jay Wimalasena, University of Tennessee Medical Center.

    Address correspondence to Dr. C. Dees, Research and Development, PhotoGen L.L.C, #1 805, 790 N. CedarBluff Road, Knoxville, TN 37923. Telephone: (423) 531-3192. Fax: (423) 458-2879. E-mail: GENASE@aol.com

    Abbreviations used: Cdk2, cyclin-dependent kinase 2; HSP, heat shock protein; PBS, phosphate-bufferedsaline; PKC, protein kinase C; RLE, rat liver epithelial (cells); TPA, 12-O-tetradecanoylphorbol-13-acetate.

    examined the ability of these two solventsto induce cyclin-dependent kinase 2 (Cdk2)activity and to hyperphosphorylate pRb 1O5.Cdk2 has been previously associated withthe promotional effects of estrogen andphosphorylates the tumor-suppressor geneproduct pRblO5 (6). We hypothesizedthat differences in kinase induction andpost translation modification of proteinslike pRblO5 might account for the appar-ent differences in carcinogenicity shown bybenzene and toluene. Hyperphosphoryla-tion of pRblO5 was examined in rat liverepithelial (RLE) and HL60 cells that hadbeen treated with benzene or toluene.Cdk2 kinase activity in cells treated withbenzene and toluene was also determined.

    Previous studies have also suggestedthat p53 DNA site-specific DNA bindingis increased in cells treated with DNA-damaging agents and chemotherapeuticagents (7). We hypothesized that the abil-ity to damage DNA might be responsiblefor the apparent differences in carcinogenicpotential of benzene and toluene. Therefore,we examined p53-DNA binding in RLEcells treated with toluene or benzene.

    Materials and MethodsCell lines examined in this study includedWB-F344 RLE cells, which were a giftfrom Dr. James Trosko of Michigan StateUniversity (1). Because RLE cells appear toproduce a wild-type p53 and pRblO5 (8),they were used for immunoprecipitation ofpRblO5, Cdk2, and p53-DNA sequence-specific binding studies. Because the HL60cells, obtained from the American TypeCulture Collection, produce mutant p53and the pRblO5 status is unknown, theywere only examined for Cdk2 activation.

    RLE cells were maintained in Richter'smedium, and HL6O cells were maintainedin RPMI 1640. Both cell lines were incu-bated at 37C in a 5% CO2 atmosphere.RLE cells were maintained with 5% (v/v)calf serum and HL60 cells in 20% fetalbovine serum. Prior to studies on Cdk2 acti-vation, the respective serum concentrationswere reduced to 0.5% (v/v) for 48 hr.

    Hyperphosphorylation of pRb 105 inRLE cells was examined by adding 0 to 2%(v/v) benzene or toluene to the medium ofthe adherent cells similar to proceduresdescribed previously (1). Briefly, pRblO5was radiolabeled using [32P]-ortho-phos-phoric acid and the cells were lysed (1).pRblO5 was immunoprecipitated (1). Totalprotein of all extracts was determined using

    Environmental Health Perspectives * Vol 104, Supplement 6 * December 1996 l1289


    a commercial BCA protein assay (PierceBiochemicals, Rockford, IL). Samples wereequalized on total protein before immuno-precipitations were performed. Anti-pRb 105 monoclonal antibody was obtainedfrom Oncogene Sciences (Manhassett,NY). Radiolabeled cell extracts were incu-bated with the antibody and protein A/Gagarose overnight. The agarose immunecomplexes were precipitated using micro-centrifugation, and then the supernatantswere removed and discarded. The agarosebeads were washed with lysis buffer andthen centrifuged. Standard denaturing gelsample buffer was added, and the immuno-precipitates examined using denaturing gelelectrophoresis followed by autoradiogra-phy. Polyacrylamide gels used were 8.0%from a commercial source (Novex Inc.,San Diego, CA). Immunoprecipitationstudies were performed similar to thekinase assay protocols except [35S]-methio-nine labeled extracts from RLE cells wereused (1). Anti-p53 (ab-1) and anti-heatshock protein (HSP) 72/73 were obtainedfrom Oncogene Sciences.

    RLE or HL60 cells were washed twicewith ice-cold phosphate-buffered saline(PBS) and lysed by the addition of coldlysis buffer (Tris 20 mM pH 7.5, NaCl250 mM, 0.1% NP-40, NaF 10 mM,sodium vanidate [NaVO] 1 mM, phenyl-methylsulfonylfluoride [PMSF] 1mM).After 15 min on ice, the lysates were cen-trifuged at 20,000g for 15 min (4C).Cdk2 was precipitated from equal amountsof cell extracts using purified rabbit anti-Cdk2 (Santa Cruz Biotechnology, SantaCruz, CA) and protein A/G agarose. Cdk2immunoprecipitates were washed (3x) withthe lysis buffer and twice with kinase buffer(Tris 40 mM, pH 7.5, MgCl2 10 mM).The immunoprecipitates were suspendedin 30 p1l of kinase buffer supplementedwith 400 pg/mi histones (type II-SS, Sigmachemical, St. Louis, MO), 5 pM ATP, 0.5pM dithiothreitol, 0.5 mM EGTA, and 5pCi y-[32P]-ATP for 20 min at room tem-perature. The reaction was stopped usinggel electrophoresis sample buffer, and thereaction products were separated on a 14%polyacrylamide gel (Novex).

    Cells were cultured in 175-cm2 flasks inRichter's medium supplemented with 0.5%calf serum. The medium was replacedbefore adding compounds for test withfresh medium without serum. Cells werethen incubated for 2 hr. Untreated controlcells were also examined. Nuclear extractsfrom the cells were prepared as described(5). Briefly, the medium was removed

    from the cells, and the monolayers werewashed with PBS, pH 7.4. Cells were lysedby the addition of 2.5 ml buffer (20%glycerol, 10 mM NaCl, 1.5 mM MgC12,0.2 mM EDTA, 1 mM dithiothreitol, 1mM PMSF, and 0.1% Triton X-100 in 20mM HEPES buffer, pH 7.6). The lysatewas centrifuged at 800g for 4 min and theresulting pellet was diluted with 3 vol of500 mM NaCl in buffer (see above) andthen incubated at 4C for 30 min with agi-tation. The mixture was centrifuged at35,000g for 10 min and the supernatantscontaining p53 were removed for immedi-ate analysis. The total protein content ofthe extracts was determined using BCAprotein assays (Pierce Biochemicals).Protein content for all samples was equal-ized before performing the binding assay.The consensus p53 binding sequencedetermined by Funk (GGACATGCC-CGGGCATGTCC) (9) was synthesized,prepared in double-stranded form, andend-labeled with [32P]-ATP. Binding reac-tions consisted of 20 pg nuclear protein,0.5 ng 32P-labeled oligonucleotide, 0.5 pgsalmon sperm DNA (Sigma) with buffer

Carcinogenic Potential of Benzene and Toluene When Evaluated
Carcinogenic Potential of Benzene and Toluene When Evaluated
Carcinogenic Potential of Benzene and Toluene When Evaluated
Carcinogenic Potential of Benzene and Toluene When Evaluated
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