Benzo[a]pyrene metabolism in cells productively infected with Simian virus 40

Chem.-BioL Interactions, 32 (1980) 111--123

111

© Elsevier/North-Holland Scientific Publishers Ltd.

BENZO[a]PYRENE METABOLISM IN CELLS PRODUCTIVELY INFECTED WITH SIMIAN VIRUS 40*

M.R. HALL and T.A. STOMINGa Department of Microbiology, School of Medical Sciences, University o f Nevada, Reno, N V 89557 and aDepartment of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30902 (U.S.A.)

(Received February 27th, 1979) (Revision received May 12th, 1980) (Accepted May 26th, 1980)

SUMMARY The metabolism of benzo[a]pyrene (BP) by cell cultures and cell-free extracts of the m o n k e y kidney cell line CV-1 was studied in uninfected and Simian virus 40 (SV40) infected cells. Metabolites formed were separated by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation techniques. The profiles of metabolites formed by uninfected and SV40 infected cells were similar except that SV40 infected cell cultures metabolized BP at an increased rate relative to uninfected cells. In addition, SV40 infected cell cultures and cell-free extracts produced an u n k n o w n c o m p o u n d which eluted between the 3-hydroxybenzo[a]pyrene and BP fractions. This material does n o t have a retention time characteristic of any of the known metabolites o f BP. Labelled BP and/or its metabolites were bound to the viral DNA and histone components of intracellular viral minichromosomes as well as the viral DNA and proteins of mature virions.

INTRODUCTION There is substantial evidence that viruses and chemicals cause tumors in several animal species, and viruses and chemicals have both been implicated as possible etiological agents of human cancers. However, investigations of *This work was supported by USPHS grants CA-23081 and CA-21481 from the National Cancer Institute, by grant NP-189 from the American Cancer Society, and by a Reno Cancer Center Research Grant. Abbreviations: BP, benzo[a]pyrene; DMSO, dimethylaulfoxide; OHBP, hydroxybenzo[a]pyrene; PAIl, polycyclic aromatic hydrocarbons; PBS, phtmphate buffered saline; p.f.u., plaque-forming units, SV40, Simian virus 40.

112 interactions between chemical carcinogens and oncogenic DNA viruses have been sporadic and heterogeneous enough in their use of different cell lines and carcinogens that few clear patterns are evident. It has been reported that replication of Herpes simplex virus types 1 and 2 [1--3], Herpesvirus saimiri [4] and SV40 [5] is inhibited by carcinogenic polycyclic aromatic hydrocarbons. In contrast to their inhibiting effects on virus replication, with few exceptions [6], chemical carcinogens appear to enhance the frequency of transformation of mammaliam cells by herpesviruses [7], adenoviruses [8--10], and SV40 [11- 1 3 ] . One of the more c o m m o n mechanisms postulated to explain enhancement of viral transformation by chemical carcinogens is that interaction of carcinogen with cellular DNA produces lesions in the DNA, thus providing sites for integration of the virus genome [7,10,13]. The studies described above cannot be directly compared with recent investigations of chemical oncogenesis in culture because no information was presented in them concerning the metabolic fate of the chemicals in question in virus infected cells. It is n o w generally accepted that chemical oncogens that are not themselves chemically reactive must be converted metabolically into reactive products [14]. With respect to PAHs such as BP, it is n o w known that they are metabolized to a variety of products including phenols, diols, quinones and glutathione conjugates [14--18]. Recent studies have shown that the 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene-9,10-oxide is the most mutagenic metabolite of BP [19--21] and it has been shown to be an ultimate carcinogenic metabolite of BP [22]. The present investigations were undertaken to determine whether BP is metabolized in SV40 virus infected cells and ultimately to determine the effects of BP metabolic intermediates on viral replicative and transforming functions. MATERIALS AND METHODS

Virus. Stock preparations of SV40 {strain RH911} were grown in CV-1 cells, a continuous line of African green m o n k e y kidney cells [23], by methods previously described [24]. The titers of these preparations ranged from 5 × 107 to 1 × 10 a plaque-forming units (p.f.u.)/ml when assayed on CV-1 monolayers. Cells. CV-1 cells were grown as monolayers in 100 mm plastic petri dishes in 10.0 ml of Eagle medium containing 10% newborn calf serum. Cells were subcultured twice weekly by trypsinization with 0.25% trypsin and were used to seed new dishes at a concentration of approx. 5 × l 0 s cells/dish in fresh medium. Chemicals. BP, purchased from Aldrich Chemical Co., San Leandro, CA, was recrystalized from ethanol and solubilized in dimethylsulfoxide {DMSO} to a final concentration of 10 mM. Stock solutions were stored at - 7 0 ° C in foil covered flasks. General labeled [3H]BP (20 Ci/mmol) purchased from Amersham/Searle, Arlington Heights, IL, was purified by passage through a

113 silica gel (100--200 mesh) column eluted with cyclohexane. BP metabolite standards consisting of trans-7,8~lihydrobenzo[a]pyrene-7,8-diol, BP-1,6dione, BP-3,6~lione, 9-hydroxybenzo(a)pyrene (9-OHBP), and 3-OHBP were generous gifts from Dr. J.K. Selkirk, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN. B P cytotoxicity. To avoid toxic concentrations of BP, its effect on the plating efficiency of CV-1 cells was determined under conditions similar to those described by Docherty et al. [6]. Confluent monolayers of CV-1 cells were dissociated with 0.25% trypsin and viable cell counts were performed by trypan blue exclusion. The cell suspension was adjusted so that approx. 400 viable cells were contained in 4.5 ml of growth medium which was pipetted into each of 54 plastic petri dishes (60 ram). To each of the series of 9 dishes, 0.5 ml of one of 4 dilutions of carcinogen was added. Controls consisted of 9 untreated dishes, and 9 dishes which received 0.5 rnl of a 1 : 100 dilution of D M S O . Trays of BP-treated cultures were covered with foil and incubated at 37°C in atmosphere containing 7 % CO2. O n day 7, the medium was removed and clones were stained for 2 rain with crystal violet. B P metabolism. BP metabolism was determined with live cells in culture and with crude sonicated cell preparations. For experiments with cells in culture, labeled BP (1.0 ~Ci/ml) was added to mock-infected or infected cells as freshly prepared solutions in D M S O . The final BP concentration was 10 -s M. Controls consisted of the selected concentrations of BP in petri dishes containing medium but no cells. After 42 h incubation at 37°C (42 h post-infection), the medium (5.0 ml) and cells from triplicate 100 m m petri dishes were removed by scraping the cells into the medium with a rubber policeman and extracted 3X with equal volumes of ethylacetate [25]. Ethylacetate extracts were pooled, dried over anhydrous sodium sulfate, evaporated to dryness and the residue (metabolites) dissolved in 75 ~I methanol for H P L C analysis [26,27] (see H P L C methods below). Estimates of the percentages of [3H]BP metabolites remaining in the aqueous phase and those extracted in the organic phase were made by dispensing 200-~I aliquots of the ethylacetate extracted medium and the ethylacetate extracts into scintillation vials, evaporating to dryness and counting by standard liquid scintillationtechniques. Cell extracts for BP metabolism experiments were prepared from mockinfected or infected cell monolayers 42 h after infection. Each culture was washed 3X with phosphate buffered saline (PBS) (0.015 M Na2HPO4; 0.0002 M KH2PO4; 0.002 M KCI; 0.08 M NaCI; p H 7.5). The cells from each petri dish (100 ram) were scraped in 2.0 ml of PBS, identical cell suspensions were pooled and centrifuged at 800 × g for 10 rain at 4°C. Each cell pellet was resuspended in 1.0 ml (10 ~ cells/ml) of ice cold hypotonic buffer (0.05 M Tris--HCl, p H 7.5) and allowed to swell for 10 min. The cell suspension was then sonicated for 10 sec at 0°C. Metabolites were formed by incubating the crude cell sonicate in a reaction mixture similar to that described by Selkirk et al. [27]. Each reaction vessel contained, in a total volume of 1.0 ml; 1--2 m g of protein sonicate, 0.55 ~ M N A D P H , 3 /~M

114 Tris--HC1 (pH 7.5) and 100 nM o f [3H]BP (spec. act., 70.6 mCi/mM) dissolved in 40 pl of methanol. Control flasks received sonicate which had been incubated at 100°C for 30 min. Flasks were incubated for 45 rain at 37°C under reduced light and the reaction was terminated b y addition of 0.5 ml of acetone. Reaction mixture was then extracted with 2.0 ml o f ethylacetate, followed by a second extraction with 1.0 ml of ethylacetate. The ethylacetate fractions were pooled and dried over sodium sulfate. The organic material was filtered and the sodium sulfate was then washed with 0.5 ml o f ethylacetate. Ethylacetate extracts were combined, evaporated to dryness with nitrogen and the residue dissolved in 75 ul of methanol for HPLC analysis. BP metabolite standards were added to each sample residue to assist in identification during HPLC analysis. In all cases, these experiments were repeated three times and each experiment was sampled in triplicate. HPLC separation o f BP metabolites was conducted on a Waters Associates Model 440 liquid chromatograph equipped with a 254 nm detector, model 6000-A solvent delivery system, model U6K universal injection system, model 660 solvent programmer and a pBondapak C~s column (4 mm I.D. × 30 cm). The column was eluted with a linear gradient of methanol and water (60% methanol/water to 80% methanol/water over 40 min) followed by a step to 100% methanol. The flow rate was maintained at 1.0 ml/min and fractions were collected at 30-sec intervals. The effluent was monitored by a 254-nm ultraviolet detector. Radioactivity was determined by pipetting a 200 ~l aliquot of each sample into TT-21 scintillation fluid (Yorktown Research) and counting in a Beckman LS-230 counter. Binding o f BP to viral components. Confluent monolayers were infected with SV40 at a multiplicity of 10 p.f.u./cell. Immediately after infection, monolayers were treated with [3H]BP (10 uCi/ml) at a final BP concentration of 10 -s M. Viral DNA and nucleoprotein complexes were extracted 42 h post-infection. Complete virions were isolated 7--8 days post-infection. Viral DNA was isolated from BP treated cells b y the Hirt selective extraction procedure [28] and purified as previously described [24]. Viral nucleoprotein complexes were extracted from BP-treated cells and purified by methods previously described in detail [29,30]. Total histones were extracted from purified viral nucleoprotein complexes with 0.5 N H2SO4 as described by Panyim et al. [31]. Mature SV40 virions were extracted from cell lysates with 1,1,2-trichlorotrifluorethane [32]. Virus was purified from the trifluoroethane extracts by the method of Meinke et al. [33] or by the method of Schaffhausen and Benjamin [34]. Virus production in control and BP-treated cells was monitored by standard SV40 plaque assay methods [35]. RESULTS

Cytotoxicity studies The effect of various concentrations of BP on the multiplication o f CV-1

115 TABLE I E F F E C T OF BP ON PLATING EFFICIENCY OF CV-1 CELLS Cultures were prepared, carcinogen treated and analyzed as described in Materials and Methods. Treatment molar concentration

Number o f clones in nine plates

Plating efficiency (%) Absolute a

Relativeb

None DMSO BP 10 -s B P 1 0 -6 B P 1 0 -~ BP 10 -s

1074 1068 1136 1065 1106 1079

29.8 29.6 31.5 29.6 30.7 30.0

100 99.4 105 99.2 104 100

a Percentage o f plated viable cells forming a clone. b Percentage o f cells forming a clone relative to untreated controls.

cells is shown in Table I. In agreement with the results of Diamond [36], CV-1 cells were not sensitive to growth-inhibition by any of the concentrations of BP which were examined. Based on these results, BP was subsequently used at the highest concentration tested, 0.01 raM. BP metabolism. The initial separation of BP metabolites was based on their extractability from an aqueous solvent, growth medium, into the organic solvent ethylacetate. Approximately 65% of the total radioactivity added could be extracted into the organic phase with samples from either infected or uninfected cell cultures. Furthermore, only 11% of the total input radioactivity could be detected in the aqueous phase of samples from infected or uninfected cells. Analyses of the BP metabolites extracted into ethylacetate from CV-1 cells and their spent culture medium as well as from CV-I crude cell-free extracts, were performed by HPLC. Figure 1 compares the metabolite profiles obtained with a synthetic mixture of metabolic standards (top), cultures of m o c k infected CV-1 cells (middle) and cultures of SV40 infected CV-1 cells (bottom). Although a synthetic metabolite mixture was added to all test metabolic extracts, they have been charted separately for clarity in presenting the metabolically produced profiles. The several quinones produced by metabolism of BP are poorly resolved by the HPLC techniques utilized in this investigation and the same is true for phenols. Therefore, since individual metabolites within these two groups cannot be identified with certainty the peaks of metabolites are identified only as diols, quinones and phenols. Several radioactive peaks (Fig. 1) emerging from the column early are within the fractions usually identified as BP~liols [17,26,27], with peak 7 cochromatographing with the 7,8-diol standards. Radioactive peaks 9, 10 and possibly 11 chromatograph within the region of quinones. Peaks 9 and 10 cochromatograph with the 1,6 quinone and 3,6 quinone standards,

116

Standards

"

'

Phenc4sr ~ ~

'

BP

CN

Mock infeded e,i

x

2

1 2

3

5

sv4o

d

4

9

I

~

,k

2; x

13

1

67 40

9 f

6O 80 Fraction Number Fig. 1. HPLC profiles of a synthetic mixture of BP and some of its metabolites, BP metabolites formed by mock infected cultures of CV-1 cells, and BP metabolites formed by S V 4 0 infected CV-1 cells. Infected and uninfected monolayers of CV-1 cells were exposed, for 42 h, to [3H]BP (10 ~Ci/ml) at a final BP concentration of 0.01 raM. BP metabolites were extracted from cells and culture medium with ethylacetate and analyzed by HPLC as described in Materials and Methods. The radioactivity plotted on this chart has been adjusted to account for non-enzymatic modification of BP determined by addition of BP to petri dishes which did not contain cells. Note the scale differences in the ordinates of the middle and bottom panels. 20

respectively. Peaks 12 and 13 c o c h r o m a t o g r a p h with m e t a b o l i t e s t a n d a r d s 9-OH-BP a n d 3-OH-BP within the p h e n o l region o f the H P L C profile. R a d i o a c t i v e m e t a b o l i t e peak 14 is u n i d e n t i f i e d at this time, h o w e v e r , it is o f interest because it o n l y appears in H P L C profiles o f m e t a b o l i t e s prod u c e d b y S V 4 0 i n f e c t e d cells {Fig. 1, b o t t o m } o r b y c r u d e e x t r a c t s o f S V 4 0 i n f e c t e d cells {Fig. 2, b o t t o m } . Also e v i d e n t f r o m these profiles, is

117 TABLE II

Q U A N T I T A T I O N OF T H E M A J O R M E T A B O L I C P R O D U C T S P R O D U C E D M E T A B O L I S M O F [3H]BP B Y CV-1 CELLS A N D CELL-FREE E X T R A C T S Products a

DPM/fraction Cell Extracts

Cells

Diols Quinones Phenols Peak 14 Metabolites as b % Total [3H]BP

BY

Mock Inf.

SV40 Inf.

Mock Inf.

SV40 Inf.

80 1200 340 0

260 5450 3800 5940

620 1510 5230 0

370 1950 3960 1470

0.05%

0.25%

0.39%

0.34%

a The data summarized in this table are derived from the metabolite profiles presented in Fig. 1 and 2. b Total metabolites were determined by summing all radioactivity eluting from the HPLC column prior to BP.

the fact that there is approx. 5× as much radioactivity in metabolites from SV40 (Fig. 1, bottom and Table II) infected cells as from m o c k infected cells (Fig. 1, middle). Figure 2 is an H P L C profile of the metabolites formed by cruded cell-free extracts of mock-infected and SV40 infected SV-1 cells.These profiles are similar to those of whole cells in that they each contain several metabolites within the diol, quinone and phenol metab~lite regions. However, they differ from the whole-cell metabolite profiles in two areas. First, the major radioactive metabolite peaks from cell-free extracts chromatograph in the phenol region of the profile rather than in the quinone region. Second, there are approximately equal amounts of total radioactivity in metabolites formed by cell-free extracts of infected and uninfected cells (Fig. 2 and Table II). B P binding to viral macromolecules. The patterns of [3H]BP binding to SV40 virus chromosomes were determined by extracting viral DNA-protein complexes from infected cells which had been treated with [3H]BP for 42 h prior to nucleoprotein complex extraction. Viral nucleoprotein complexes were then purified as previously described and analyzed by velocity gradient centrifugation in 5--20% sucrose gradient (Fig. 3). The peak of [3H]BP labelled material in fractions 12--17 has a sedimentation velocity (63S) well within the previously published range for SV40 nucleoprotein complexes [24]. These calculations were based on the sedimentation properties of [14C]thymidine labelled viral D N A (21S) included as a marker. Since SV40 virus NPCs contain viral D N A and protein (primarily histone) in approximately the same proportion as eukaryotic chromatin [37], it was of interest to determine the proportion of [3H]BP bound to each component. Therefore, histones and viral D N A were extracted from

118 S~dards

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BP

,78-D~

Q~n0o~

<[

I

t

1.5

10

5 ,,,

11 I 2

5

9~II~

1

10

~)

40

60

80

100

Fig. 2. HPLC profiles of a synthetic mixture of BP and some of its metabolites, BP metabolites formed by cell-free extracts of mock infected CV-1 cells and BP metabolites formed by cell-free extracts of SV40 infected CV-1 cells. Sonicated extracts of CV-1 cells were prepared 42 h post-infection. Metabolites were formed by incubating the crude sonicate (1--2 mg/ml) with [~H]BP as described in Materials and Methods. Metabolites were extracted from reaction mixtures with ethylacetate and analyzed by HPLC as described in Materials and Methods. The radioactivity plotted on this chart has been adjusted to account for non-enzymatic modification of BP determined by incubation of BP with heat inactivated extract (100°C, 30 min). s e p a r a t e a l i q u o t s o f t h e p u r i f i e d NPC p r e p a r a t i o n a n d t h e i r specific activities o f b o u n d [3H]BP d e t e r m i n e d . T h e averages o f t h r e e d e t e r m i n a t i o n s p e r c o m p o n e n t w e r e 3.1 D P M / u g o f D N A a n d 32.1 D P M / u g o f h i s t o n e . Viral D N A e x t r a c t e d f r o m [ 3 H I B P t r e a t e d i n f e c t e d cells b y t h e H i r t p r o c e d u r e or p h e n o l e x t r a c t e d f r o m p u r i f i e d virions h a d specific activities similar t o t h a t o f n u c l e o p r o t e i n c o m p l e x e s , 2.7 D P M / ~ g a n d 3.0 D P M / ~ g , r e s p e c t i v e l y . In a d d i t i o n , t o t a l viral p r o t e i n s isolated f r o m p u r i f i e d virions h a d a specific a c t i v i t y ( 3 5 . 0 DPM//Jg) similar t o t h a t o f n u c l e o p r o t e i n c o m p l e x histones.

119

20

4 m

,•15 X

'i IO

2i

,,, 5

I

I0 20 50 40 FRACTION NUMBER Fig. 3. Velocity sedimentation analysis of [3H]BP-modified SV40 nucleoprotein complexes in 5--20% sucrose gradients. Confluent monolayem of CV-1 cells were infected with SV40 and immediately treated with [3H]BP. Afrer 42-h incubation, viral nucleoprotein complexes were isolated as previously described [29,30]. Purified nucleoprotein complexes were mixed with [:4C]thymidine-labeiled SV40 DNA and centrifuged as described [24 ]. Sedimentation is from right to left.

Infectivity of fJH]BP modified virions. The replicative ability of SV40 virions grown in the presence of 0.01 M BP and 0.5?0 DMSO was determined by plaque assay on monolayers of CV-1 cells. The results of experiments with three independently produced pools o f virus are summarized in Table III. As previously discussed (Materials and Methods) control preparations of SV40 virus had titers of from 5 X 107 to 1 × 10 s TABLE III EFFECT OF BP ON SV40 VIRUS REPLICATION IN CV-1 CELLS. Confluent monolayem, infected with SV40, were treated with selected concentrations of BP and incubated for 7 days. The culture medium, containing SV40, was clarified by centrifugation and virus titer was determined by standard plaque assay techniques [ 32 ]. Virus growth conditions

BP concentration (M)

Virus titer p.f.u./ml x 10 ~

Control DMSO (0.5%) DMSO (0.5%)

-1 x I 0 -s

8.0 -* 2.7" 7.0 -~ 3.0 2.2 +- 2.5

DMSO (0.5%) DMSO (0.5%) DMSO (0.5%)

1 x I 0 -6 1 x 10 -~ 1 x 10 -s

7.8 -* 2.7 8.1 ~- 2.0 '/.9 ~ 3.1

a Mean

± S.D.

120 p.f.u./ml. Growth in the presence of 0.5% DMSO did not effect productive yield or infectivity of SV40 virions in CV-1 cells. However, virus yield from cells treated with 10 -s M BP was inhibited by approx. 75%. In addition, the infectivity of virus produced in the presence of 10 -5 M BP was reduced relative to that of controls. The BP concentration o f 10 -5 M was the highest concentration tested because BP was n o t soluble in our growth medium at higher concentrations. Purified control virus preparations yielded approx. 1.7 × l 0 s p.f.u./1.0 O.D.260 equivalent while purified 10 -5 M BP grown virions yielded only 3.4 × 10 ~ p.f.u./1.0 O.D.260 equivalent. DISCUSSION Over a period o f approx. 20 years, several laboratories have reported analyses of the effects of chemical carcinogens on the replicative and transforming potential of oncogenic DNA viruses. Although there are exceptions [6], in general, carcinogenic chemicals appear to enhance transformation [7--13] and inhibit replication [1-.-5] of DNA viruses. These effects were assumed to be due to interaction o f the chemicals with viral and cellular macromolecules, particularly DNA. However, it is now known that chemical carcinogens such as MCA and BP must be metabolically 'activated' to c o m p o u n d s which bind to macromolecules. Until now, these significant aspects of chemical oncogenesis have not been approached in investigations of the effects of carcinogenic chemicals on oncogenic DNA viruses. The results of this study demonstrate that CV-1 cells infected with SV40 are capable of metabolizing the polycyclic hydrocarbon BP. That uninfected CV-1 cells are capable o f metabolizing BP is not unexpected since Diamond [36] reported that CV-1, as well as other primate cell lines, metabolized BP to water soluble derivatives. Significantly, the HPLC patterns of BP metabolites formed by CV-1 cells correlate well with previous reports of BP metabolism and 'activation' by tissues of several mammalian species [16--18]. The peaks in the diol region o f the chromatogram are particularly interesting since the 7,8-diol is an intermediate in the metabolic formation o f the p o t e n t carcinogen, 7,8-OHBP-9,10-oxide [20]. In addition, the presence of BP-phenols in CV-1 extracts is of interest because most phenols are believed to be non-enzymatically derived from metabolically produced epoxides which are precursors to the carcinogenic and mutagenic BP metabolites [20,38,39]. Many previous reports have suggested that the enhancing effects of chemical carcinogens on transformation by DNA viruses are due to changes in cell susceptibility to viral transformation [7--13]. However, the finding that BP metabolites are b o u n d to intracellular precursors of SV40 and to the DNA and proteins of mature virions indicates that the possibility of direct effects of b o u n d metabolites on virus functions must be considered. This is particularly relevant in view of the very high mutagenic potential of BP metabolites [21,40].

121 Several laboratories have reported that SV40 [41,42] and Herpesviruses [41,43,44], made incapable of multiplying, expressed viral gene products and gave rise to cell lines. Some of these cell lines appeared transformed and were oncogenic to experimental animals [41]. Thus, it seems conceivable that growth in the presence of BP might lead to production of virus with decreased potential for replication but increased potential for transformation of even normally permissive cells. ACKNOWLEDGEMENTS

The authors acknowledge the excellent technical assistance provided by Ms. Brenda Kozyra and Ms. Betty Schleh. REFERENCES 1 E. DeMaeyer and J. DeMaeyer-Guignard, Effects of polycyclic aromatic hydrocarbons on viral replication: similarity to actinomycin D, Science, 146 (1964) 650. 2 J.J. Docherty, R.V. Goldberg and F. Rapp, Differential effect of 7,12-dimethylbenz(a)anthracene on infectivity of herpes simplex virus type 2, Proc. Sod. Exp. Biol. Med., 136 (1971) 328. 3 R.J. Goldberg, J.J. Docherty and F. Rapp, Inhibition of synthesis of Herpes Simplex Virus deoxyribonucleic acid by a carcinogenic polycyclic aromatic hydrocarbon, Proc. Soc. Exp. Med., 140 (1972) 1054. 4 F.G. Pearson and J.S. Beneke, Inhibition of Herpesvirus saimiri replication by phosphonoacetic acid, benzo(a)pyrene, and methylcholanthrene, Cancer Res., 37 (1977) 42. 5 G.T. Chang, R.G. Harvey, W-T. Hsu and S.B. Weir, Inactivation of SV40 replication by derivatives of benzo(a)pyrene, Biochem. Biophys. Res. Commun., 88 (1979) 688. 6 J.J. Docherty, P.P. Ludovici and G.T. Schloss, Inhibitory effect of carcinogenic aromatic hydrocarbons on transformation of 3T3 cells by SV40, Proc. Soc. Exp. Biol. Med., 140 (1972) 969. 7 M.K. Howett, A.E. Pegg and F. Rapp, Enhancement of biochemical transformation of mammalian cells by herpes simplex virus following nitrosomethylurea treatment, Cancer Res., 39 (1979) 1041. 8 B.C. Casto, Enhancement of adenovirus transformation by treatment of hamster cells with ultraviolet light, DNA base analogs, and dibenz (a,h)-anthracene, Cancer Res., 33 (1973) 402. 9 B.C. Casto, W.J. Pieczynski and J.A. DiPaolo, Enhancement of adenovirus transformation by treatment of hamster cells with carcinogenic polycyclic hydrocarbons, Cancer Res., 33 (1973) 819. 10 B.C. Casto, W.J. Pieczynski and J.A. DiPaolo, Enhancement of adenovirus transformation by treatment of hamster embryo cells with diverse chemical carcinogens, Cancer Res., 34 (1974) 72. 11 L. Diamond, R. Knorr and Y. Shimizu, Enhancement of simian virus 40-induced transformation of Chinese hamster embryo cells by 4-nitroquinoline 1-oxide, Cancer Res., 34 (1974) 2599. 12 K. Hirai, V. Defendi and L. Diamond, Enhancement of simian virus 40 transformation and integration by 4-nitroquinoline 1-oxide, Cancer Res., 34 (1974) 3497. 13 G.H. MUo, J.R. Blakeslee, R. Hart and D.S. Yohn, Chemical carcinogen alteration of SV40 virus induced transformation of normal human cell populations in vitro, Chem.Biol. Interact., 22 (1978) 185. 14 C. Heidelberger, Chemical carcinogenesis, Ann Rev. Biochem., 44 (1975) 79.

122 15 E. Boyland and P. Sims, The metabolism of benz(a)anthracene, Biochem. J., 91 (1964) 493. 16 P. L. Grover, A. Hewer and P. Sims, Formation of K-region epoxides as microsomal metabolites of pyrene and benzo(a)pyrene, Biochem. Pharmaco.., 21 (1972) 2713. 17 S.K. Yang, J.K. Selkirk, E.V. Plotkin and H.V. Gelboin, Kinetic analysis of the metabolism of benzo(a)pyrene to phenols, dihydrodiols, and quinones by highpressure liquid chromatography compared to analysis by aryl hydrocarbon hydroxylase assay, and the effect of enzyme induction, Cancer Res., 35 (1975) 3642. 18 T.H. Zytkovicz, H.L. Moses and T.C. Spelsberg, The binding of benzo(a)pyrene and N-methyl-N'-nitro-N-nitrosoguanidineto subnuclear fractions of AKR mouse embryo cells in culture, Int. J. Cancer, 20 (1977) 408. 19 R.F. Newbold and P. Brookes, Exceptional mutagenicity of a benzo(a)pyrene diol epoxide in cultured mammalian cells, Nature, 261 (1976) 52. 20 D.R. Thakker, H. Yagi, A.Y.H. Lu, W. Levine, A.H. Cooney and D.M. Jerina, Metabolism of benzo(a)pyrene: conversion of (_+)-trans-7, 8
123 35 J.E. Cleaver, Plaque formation with simian virus 40: enhancement by dimethyl sulfoxide, J. Virol., 14 (1974) 1607. 36 L. Diamond, Metabolism of polycyclic hydrocarbons in mammalian cell cultures, Int. J. Cancer, 8 (1971) 451. 37 C. Cremisi, P.F. Pignatti, O. Croisse__nt and M. Yaniv, Chromatin-like structures in polyoma virus and simian virus 40 lyric cycle, J. Virol., 17 (1976) 204. 38 R.I. Freudenthal, A.P. Leber, D. Emmeding and P. Clarke, The use of high pre~ure liquid chromatography to study chemically induced alterations in the pattern of benzo(a)pyrene metabolism, Chem.-Biol. Interact., 11 (1975) 449. 39 S.K. Yang, P.P. Roller and H.V. Gelboin, Enzymatic mechanims of benzo(a)pyrene conversion to phenols and diols and an improved highpreuure liquid chromatographic separation of benzo(a)pyrene derivatives, Biochemistry, 16 (1977) 3680. 40 A.W. Wood, P.G. Wislocki, R.L. Chang, W. Levin, A.Y.H. Lu, H. Yagi, O. Hernandez, D.M. Jerina and A.H. Conney, Mutagenicity and cytotoxicity of benzo(a)pyrene benzo-ring epoxides, Cancer Res., 36 (1976) 3358. 41 F. Rapp, J.H. Li and M.A. Jerkofsky, Transformation of mammalian cells by DNAcontaining viruses following photodynamic inactivation, Virology, 55 (1973) 339. 42 N.H. Seemayer, K. Hirai and V. Defendi, Analysis of minimal functions of simian virus 40. I. Oncogenic transformation of syrian hamster kidney cells in vitro by photodynamically inactivated SV40, Int. J. Cancer, 12 (1973) 524. 43 R.G. Hughes and W.H. Munyon, Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation, J. Virol., 16 (1975) 275. 44 J.C.M. MacNab, Transformation of rat embryo cells by temperatureserudtive mutants of herpes simplex virus, J. Gen. Virol., 24 (1974) 143.