Environmental and Molecular Mutagenesis 19:156160 (1992)

Nitrobenzo[a]Pyrene-Induced DNA Amplification in SV40-Transformed Chinese Hamster Embryo Cells Robin E. Neft, Amy 1. Roe, Henry M. Schol, Peter P. Fu, Roberta A. Mittelstaedt, and Daniel A. Casciano Division o f Genetic Toxicology (R.E.N., A.L.R., H.M.S., R.A.M., D.A.C.) a n d Division o f Biochemical Toxicology (P.P. F.), Food a n d Drug Administration, National Center for Toxicological Research, Jefferson, a n d Department o f Pharmacology a n d Toxicology, Department o f Biochemistry, University of Arkansas for M e d i c a l Sciences, Little Rock, (D.A. C.), Arkansas Nitrobenzo[a]pyrenes (NBaPs) are ubiquitous environmental pollutants that produce mutations in Salmonella typhimurium and Chinese hamster ovary cells. In this study, 1-, 3-, and 6-NBaP induced amplification of SV40 DNA sequences in an SV40-transformed Chinese hamster embryo cell line which is sensitive to DNA amplification by various known carcinogens. Of the

three isomers, 3-NBaP produced the highest level of gene amplification, which was 4.8 relative to untreated controls at a dose of 5 pg/ml. Considering the relationship between gene amplification and tumorigenesis, it seems prudent to carry out a more exhaustive analysis of the carcinogenic potential of these agents.

Key words: mutagenesis, C060 cells, viral DNA amplification

INTRODUCTION Nitrated polycyclic aromatic hydrocarbons (nitro PAHs) are ubiquitous pollutants that are formed when PAHs, nitrogen oxides, and acids are present simultaneously in the environment and by nitration of PAHs during combustion [Pitts et al., 19781. The nitro PAHs I - , 3-, and 6-nitrobenzo[alpyrene (NBaP) are produced in model atmospheres containing nitrogen oxides, benzo[a]pyrene (BaP), and nitric acid [Pitts et al., 19781. NBaPs are found in urban air particulates and particulates generated by diesel and gasoline engines and wood-burning stoves [Jager, 1978; Pitts et a]., 1982; Schuetzle, 19831. Several studies have shown that I - and 3-NBaP are direct-acting mutagens in the Sulmonellu reversion assay and all three isomers are mutagenic in the presence of exogenous metabolic activation [Hass et al., 1986a,b; Heflich et al., 19891. In the CHO/HGPRT assay, 1- and 3-, and to a lesser degree, 6-NBaP produced positive mutagenic responses in the presence of S9 metabolic activation [Hass et al., 1986b; Heflich et al., 19891. In addition, 3-NBaP is a potent inducer of aneuploidy in CHO-KI-BH, cells "eft et al., 19901. Although many studies have evaluated the in vitro genotoxicity of these compounds, only 6-NBaP has been tested for carcinogenicity. It was negative in the mouse skin assay [El-Bayoumy et al., 19821, but positive in the newborn mouse assay (Wislocki et al., 1985, 1986). Despite the fact that numerous studies have examined the mutagenicity of NBaPs in cells in culture, molecular anal0 1992 Wiley-Liss, Inc.

ysis of cells exposed to NBaPs has not been performed. Considering the role that gene amplification may play in carcinogenesis [Stark et al., 1989; Schimke, 19841, it is of interest to study the effects of NBaPs in a model in vitro system for DNA amplification. The C 0 6 0 cell is an SV40-transformed Chinese hamster embryo cell. Several investigators have shown that many mutagens/carcinogens, including PAHs such as BaP and 7,12-dimethylbenz[a]anthracene (DMBA), can induce SV40 DNA synthesis in C 0 6 0 cells [Lavi, 1981; Lavi and Etkin, 1981; Kleinberger et al., 1988; Burkle, 1989; Pool et al., 19891. Therefore, the purpose of this study was to examine the effects of NBaPs on DNA amplification in C 0 6 0 cells. In addition, although there is some evidence [Lavi and Etkin, 19811 that C 0 6 0 cells can metabolize hydrocarbons such as BaP, it was of interest to determine whether exogenous (S9-mediated) metabolic activation could enhance the response of C 0 6 0 cells to NBaP exposure.

MATERIALS A N D METHODS Chemicals I - , 3-, and 6-NBaP were prepared and purified by a procedure previously described [Chou et al., 19841 which Received May 18, 1990: reviscd and accepted September 5 . 1991. Address reprint requests to Robin E. Neft, Divi\ion of Genetic 'Toxicology, Food and Drug Administralion. National Center for Toxicological Research. Jefferaon, AK 72079-9502.

Nitrobenzo[a]pyrene-Induced D N A Amplification

efficiently provides products free from contamination by isomers. Purity was confirmed by high performance liquid chromatographic (HPLC) analysis employing a Vydac ODS column (6.2 X 250 mm) eluted with methanol-water (911; v/v) at a flow rate of 1 .O ml1min. Under these conditions, I - , 3-, and 6-NBaP eluted at 9.4, 9.7, and 8.4 min, respectively. Their structures were confirmed by UV-visible absorption and mass spectroscopy. Their purities were 99+% based on HPLC analysis. Cells

C060 cells (kindly provided by Dr. S. Lavi, Tel Aviv, Israel) were maintained at 37"C, 5% CO,, in Dulbecco's modified Eagle medium (DMEM), supplemented with L-glutamine, penicillin (100 Uiml), streptomycin (100 pg/ ml), and 10% fetal calf serum (all purchased from Gibco). Treatment

Cells (5 x 10') were seeded into T75 tissue culture flasks (Nunclon). Twenty-four hours later the cells were treated with 1 or 5 pgiml I-, 3-, or 6-NBaP or dimcthyl sulfoxide (DMSO) (vehicle control) or 0.1 pgiml DMBA (positive control) with or without S9 (protein concentration: 0.4 mg/ml) metabolic activation in serum-free DMEM for 5 hours. The concentration of S9 used in these studies is optimum for the metabolic activation of I - , 3-, and 6-NBaP to mutagens in CHO cells [Hass et al., 1986bI. S9 was obtained from the livers of Aroclor-induced male SpragueDawley rats by the procedure of Ames et al. L197.51. Following exposure, the cells were rinsed with phosphatebuffered saline (PBS) and 15 ml of fresh DMEM were added to each flask. At 4 days post-treatment, DNA was isolated from the cells according to the method of Beland et al. I 1984 I. All chemicals were assayed on two separate occasions. Cell Survival

Cell survival was determined by relative cloning ability 24 hr after exposure of cells to 1 or 5 pgiml I-, 3 - , or 6-NBaP, DMSO, or DMBA according to the methods of Heflich et al. [1982]. Each chemical was assayed on three separate occasions. Slot Blotting

Each DNA sample (0, 1 , and 5 pg) was applied in duplicate to nylon membranes (Hybond-N, Amersham) using a Bio-Rad Bio-Dot SF slot blot apparatus. The nylon membranes were pre-hybridized in 25 mM KH,PO,, pH 7.4, 5 X SSC, 5 X Denhardt's solution, 50 pg/ml heatdenatured salmon sperm DNA, 50% formamide, and 1% sodium dodecyl sulfate (SDS) for 2 hr at 42°C. The slot blots were hybridized overnight in 10% dextran sulfate at 42°C with an SV40 DNA probe (Bethesda Research Laboratories)

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labeled with [(-w-'2P]dCTP (3,000 Ci/mmol, ICN Biomedical) to a specific activity of 2 x 10' c p d p g using a random primed DNA labeling kit (Boehringer Mannheim). The SV40 probe consisted of the entire, BamHl linearized SV40 genome. The slot blots were washed successively in 2 X SSC-O.l% SDS at 42"C, 0.2 X SSC-0.1% SDS at 55"C, and two changes of 0.2 X SSC-0.1% SDS at 42°C. Autoradiography was performed by exposure to Kodak XAR-5 film with intensifying screens at -70°C for 1-5 days. Following this procedure, the slot blots were stripped and reprobed with 32P-labeled human p-actin cDNA [Ponte et al., 19841 (a gift of Dr. D. Peffley, Chicago, IL). The resulting hybridization signals were scanned with a Zeineh scanning densitometer (model SLR-2D/lD). The absorbance values from P-actin-probed slot blots were not related to cell treatment. In addition, the range of absorbance values from P-actin-probed slots containing DNA from exposed cell cultures was small compared to the range of values from SV40-probed blots. Therefore, it appears that p-actin DNA sequences are not amplified following exposure of C060 cells to NBaPs and they can be used as an internal control for the amount of cellular DNA bound to the membranes. Amplification factors were determined (using slots containing I p g C060 DNA) by dividing the integrated absorbance values from exposed C060 cells by absorbance values from DMSO controls and multiplying these results by a correction factor for the exact amount of C060 DNA in each slot as determined from the P-actin-probed membranes.

RESULTS Slot blot autoradiograms from membranes (from two separate experiments) probed with SV40 DNA and reprobed with p-actin DNA are presented in Figure 1. The amount of amplification of SV40 DNA sequences relative to untreated controls and percent relative survival are presented in Table I. Little or no cytotoxicity was observed in C060 cells exposed to 1 or 5 pgiml 1-, 3 - , or 6-NBaP. At doses higher than 5 pgiml 1-, 3-, and 6-NBaP began to precipitate out of the tissue culture medium. Therefore, higher doses of these agents could not be tested. 1-, 3-, and 6-NBaP produced amplification that was not, in most cases, dose-dependent at concentrations of 1 and 5 pgiml. In addition, there was little change in the amount of amplification induced by all three isomers when S9 mix was present during exposure (data not shown). Furthermore, in most cases, 3-NBaP produced slightly greater amounts of amplification of SV40 DNA sequences than 6-NBaP at both doses tested. Levels of amplification in 3-NBaP-exposed C060 cell cultures reached 4.8 relative to untreated controls at a concentration of 5 pg1ml. I-NBaP induced the smallest amount of DNA amplification; however, overall large differences in the amount of amplification of SV40 DNA sequences in cells exposed to I - , 3-, and 6-NBaP were not observed.

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1

EX PER1MENT

EXPERIMENT

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PROBE

B -Acti n

SV 40

B-Actin

SV 40

a

a m

zI 'Dm

-i z

1

-

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0

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1

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Fig. 1. Autoradiograms of "P-labeled SV40 DNA probed slot blots containing 0 , I , and 5 pg DNA isolated from C060 cells treated with I or 5 pgiml I-, 3-, or 6-NBaP, DMSO (vehicle control), or 0. I pgiml DMBA (positive control) and autoradiograms of the same slot blots reprobed with '*P-labeled human p-actin DNA (only slots with I pg C060 DNA are shown)

DISCUSSION In the present study, 1-, 3-, and 6-NBaP generated amplification of SV40 DNA sequences in C 0 6 0 cells. Although previous studies [Lavi and Etkin, 1981; Pool et al., 19891 indicate that PAHs induce gene amplification in C060 cells, this is the first report indicating that nitro PAHs can do so. 3-NBaP was the most effective inducer of DNA amplification in C060 cells although large differences were not detected in the amount of amplification induced by any of the three isomers tested. It is interesting that 3-NBaP induces both aneuploidy "eft et al., 19901 and DNA amplification in Chinese hamster cells in light of the fact that Ottaggio et al. [1988] has shown that Chinese hamster cells with amplified CAD (carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene DNA sequences also exhibit aneuploidy . These results correlate with the findings of mutagenesis studies in which 3-NBaP was the most mutagenic of the three isomers [Hass et al., 1986bI. It is also interesting that 6-NBaP was weakly mutagenic in the CHO/HGPRT assay only in the presence of S9.

However, in C 0 6 0 cells 6-NBaP induced similar levels of amplification with and without S9 metabolic activation. It is likely that C060 cells are capable of ring oxidation and/or nitroreduction since PAHs induce amplification in these cells without exogenous activation [Lavi and Etkin, 19811, and it has been shown that 1-, 3-, and 6-NBaP are ring-oxidized to mutagenic compounds in the presence of rat liver microsomes and S9 [Chou et al., 1985, 1986; Thomton-Manning et al., 19881. It has been suggested that 1-, 3-, and 6-NBaP are metabolized to corresponding nitroso-BaPs [Heflich et al., 19891 because these compounds are more mutagenic than the parent nitro-BaPs in Salmonella typhirnuvium and CHO cells [Fu et al., 1988; Heflich et al., 1989; Thornton-Manning et al., 19891, but the capacity of C060 cells to perform nitroreduction is presently unknown. Unfortunately, the relationship of amplification of SV40 DNA sequences in C060 cells to DNA amplification in normal (untransformed) cells is not known. However, several studies [Lavi and Etkin, 1981; Kleinberger et al., 1988; Pool et al., 19891 have shown that C060 cells can detect the ability of various classes of mutagens/carcinogens to induce DNA amplification which may play a role in the

Nitrobenzo[a]pyrene-Induced DNA Amplification

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TABLE 1. Amplification Factors and Cell Survival Obtained at 4 Days Post-exposure of C060 Cells to 1 or 5 bg/ml 1-, 3-, or 6-NBaP Concentration Chemical

DMSO 1-NBaP

3-NBaP 6-NBaP