- Email: [email protected]
Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein–Barr virus
Toxicology 133 (1999) 35 – 42
Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein–Barr virus Hal B. Jenson a,b,*, Patty Heard a, Mary Pat Moyer b,c a
Department of Pediatrics, The Uni6ersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dri6e, San Antonio, TX 78284 -7811, USA b Department of Microbiology, The Uni6ersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dri6e, San Antonio, TX 78284 -7811, USA c Department of Surgery, The Uni6ersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dri6e, San Antonio, TX 78284 -7811, USA Received 23 September 1998; accepted 12 December 1998
Abstract Numerous chemical tumor promoters induce latent Epstein – Barr virus (EBV) to active replication. The effect of smokeless tobacco purified products N-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK), benzo(a)pyrene (BaP), and smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf tobacco) was tested for induction of latent EBV in Raji cells using fluorescence-activated cell sorter flow cytometry detection of the restricted component of EBV early antigen (EA-R). Concentrations of smokeless tobacco purified products or preparations were used that have carcinogenic effects in animal cell lines. There was no discernible effect for the 6–7-day duration of treatment on viability of Raji cells, or on induction of latent EBV in Raji cells. Results were comparable using paraformaldehyde- or acetone-fixed cells. There does not appear to be an in vitro effect of smokeless tobacco constituents on EBV-infected lymphocytes that may contribute to development of oral cancers. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Smokeless tobacco; Epstein–Barr virus; Virus induction; 12-O-tetradecanoyl phorbol-13-acetate; n-Butyrate; Fluorescence-activated cell sorter flow cytometry
1. Introduction
* Corresponding author. Present address: Department of Pediatrics, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7811, USA. Tel.: +1-210-5675301; fax: +1-210-5676305. E-mail address: [email protected] (H.B. Jenson)
Epstein–Barr virus (EBV) is associated with certain carcinomas, including nasopharyngeal carcinoma and Burkitt’s lymphoma (Raab-Traub, 1996), and is causally associated with oral hairy leukoplakia (Sciubba et al., 1989). EBV is shed intermittently from the oropharynx and can be
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 0 3 - 7
36
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
cultured from approximately 10 – 30% of immunocompetent persons (Chang et al., 1973) and from 50 – 100% of immunocompromised persons (Chang et al., 1978; Lucht et al., 1995). Chemicals known to induce replication of latent EBV infection in lymphocytes include: phorbol esters, including 12-O-tetradecanoyl phorbol-13acetate (TPA) (zur Hausen et al., 1978), a direct activator of protein kinase C (Hayashi, 1992); n-butyrate (Kallin et al., 1979; Luka et al., 1979); halogenated pyrimidines, including 5-iododeoxyuridine (Hampar et al., 1971; Gerber, 1972); and nitrosamines (Bouvier et al., 1991). Chemical induction by TPA (zur Hausen et al., 1978) leads to lytic EBV infection with a peak effect between 1 and 4 days (Sairenji et al., 1984; Boos et al., 1987; Nutter et al., 1987). The combination of EBV infection and tobacco may contribute to an increased incidence of oral cancers (D’Costa et al., 1998). Smokeless tobacco contains numerous chemicals with biologic activity and has been associated with numerous oral and pharyngeal cancers (Hoffmann et al., 1994; Gupta et al., 1996; Hoffmann and Djordjevic, 1997). The most abundant carcinogens in smokeless tobacco are the tobacco-specific nitrosamines, two of which, N-nitrosonornicotine (NNN) and 4 - (N - methyl - N - nitrosamine) - 1 - 3 - pyridinyl) - 1butanone (NNK), have been shown to increase longevity of mucosal cells in vitro with growth and morphologic changes suggestive of cell transformation (Murrah et al., 1993), and to induce benign and malignant tumors at various sites in mice, rats, and hamsters (Hecht et al., 1986; Hoffmann et al., 1991). Both NNN and NNK have been shown to pyridyloxobutylate DNA, and NNK has also been shown to methylate DNA (Murphy et al., 1990). Benzo(a)pyrene (BaP) is another carcinogenic compound found in smokeless tobacco (Obi and Billett, 1991). The Raji cell line, a human EBV genome-positive Burkitt’s lymphoma cell line, harbors approximately 50–60 EBV genome equivalents per cell (Nonoyama and Pagano, 1972; Pritchett et al., 1976). This non-producer cell line is particularly informative in the study of activation of latent EBV because of an intrinsic block after expression of the early antigens (EAs) that completely in-
hibits progression to viral DNA synthesis and late EBV gene expression (Ooka et al., 1986; Tan et al., 1986). The EA complex consists of several viral proteins expressed within the cell and is divided into two components, diffuse (EA-D) and restricted (EA-R), based on cellular localization (after fixation) and susceptibility to denaturation: EA-D is found in both the nucleus and cytoplasm, and is stable in acetone, methanol and ethanol; EA-R is found only in the cytoplasm, and is stable in acetone but denatured by methanol or ethanol (Henle et al., 1970). Latent EBV infection in the Raji cell line can be induced with TPA or n-butyrate, and can be measured by the determination of EBV early antigen (EA) production using fluorescence-activated cell sorter (FACS) flow cytometry (Jenson et al., 1998). The high prevalence of EBV infection, lifelong persistence after primary infection with replication in the oral cavity, and the carcinogens identified in smokeless tobacco and the association with oral cancers, suggest a possible cocarcinogenic interaction between EBV and smokeless tobacco. To determine the possible effect of smokeless tobacco on latent EBV, we measured virus induction in Raji cells after treatment with smokeless tobacco purified products (NNN, NNK, and BaP alone and in combination with n-butyrate (Ito et al., 1981) and tobacco extracts (dry snuff, moist snuff, and loose leaf), and compared these results to chemical induction by the tumor promoters TPA and n-butyrate, which are known to induce latent EBV.
2. Materials and methods
2.1. Smokeless tobacco extracts The tobacco extracts were prepared using standardized procedures (Murrah et al., 1993). Ten grams of either reference dry snuff, moist snuff, or loose leaf tobacco (Smokeless Tobacco Research Products, University of Kentucky, Lexington, KY) were briefly homogenized in 100 ml distilled, deionized H2O and extracted overnight at room temperature on an orbital shaker. After extrac-
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
tion, the solutions were centrifuged at 1800 × g for 30 min, and then centrifuged twice at 25000 × g for 30 min. The final supernatant was adjusted to pH 7.2–7.4 and sterile filtered through a 0.02mm syringe filter (Nalgene, Rochester, NY). The stock solution was designated as a 10% aqueous solution.
2.2. Raji cell line The Raji lymphoid cell line (ATCC CCL 86) is a human EBV-positive cell line derived from a Burkitt’s lymphoma (Pulvertaft, 1965; Epstein et al., 1966). The cells were maintained in RPMI 1640 medium (Cellgro, Mediatech, Herndon, VA) containing 10% fetal bovine serum (Hyclone, Logan, UT) with 100 U/ml of penicillin (Cellgro), 100 U/ml of streptomycin (Cellgro), 2.5 mg/ml of amphotericin B (Cellgro), and 2 mM glutamine (Cellgro) at 37°C in a humidified atmosphere with 5% CO2.
2.3. Chemical treatments Raji cells were suspended in fresh medium at 5 × 105 cells/ml and treated with one of the following chemicals: 1 mg/ml NNN (Midwest Research Institute, Kansas City, MO); 1 mg/ml NNK (Midwest Research Institute); 1 mg/ml BaP (Midwest Research Institute); 2% aqueous solution dry snuff; 2% aqueous solution moist snuff; 2% aqueous solution loose leaf tobacco (Smokeless Tobacco Research Products, University of Kentucky, Lexington, KY); 20 ng/ml TPA (Sigma, St. Louis, MO); or 4 mM n-butyric acid (Sigma). Combination treatments included NNN, NNK, BaP and TPA, each with n-butyrate (at the same concentrations used individually). The cells were harvested for analysis at 2, 4 – 5, and 6 – 7 days after treatment
2.4. Cell 6iability Absolute cell counts per milliliter and the proportion of viable cells were measured by trypan blue exclusion and by FACS flow cytometry at 2, 4 – 5 and 6–7 days after initiation of treatment. Cell viability was determined in separate aliquots
37
by trypan blue exclusion of 50 ml of cell suspension added to 0.4% trypan blue stain (GIBCO BRL, Grand Island, NY) at a 1:2 dilution. Dead cells were measured by permeabilization using propidium iodide fluorescence through a 630/22nm bandpass filter using a FACStar Plus (Becton Dickinson Immunocytometry Systems, San Jose, CA) flow cytometer with an argon-ion laser producing 200 mW of 488-nm light for excitation (Jenson et al., 1998)
2.5. Flow cytometry Cells were fixed and stained for EA using an adaptation of fixation that allows for flow cytometry analysis (Jenson et al., 1998). Cells were washed three times with phosphate buffered saline (PBS), fixed by resuspension in 1 ml of either acetone (EM Sciences, Gibbstown, NJ) or 2% paraformaldehyde (Sigma) with 0.1% Triton X (Sigma) at 4°C for 5 min (Lidin et al., 1993), and washed three times with a FACS buffer (PBS containing 10% goat serum albumin). EA-R was detected using mouse IgG2A monoclonal antibody against EA-R (Advanced Biotechnologies, Columbia, MD). The cells were stained with 20 ml of anti-EA-R antibody for 30 min at room temperature, rinsed three times with FACS buffer, and counterstained for 30 min at room temperature with goat anti-mouse IgG (heavy and light chain)–FITC conjugated antibody (Tago, Burlingame, CA) diluted 1:500 with 0.001% Evans blue in PBS. The cells were rinsed three times with FACS buffer and were resuspended in 0.5 ml of FACS buffer for FACS analysis. FACS flow cytometry was performed using a FACStar Plus (Becton Dickinson) flow cytometer with an argon-ion laser producing 200mW of 488-nm light for excitation. The fluorescence from the FITC (for EA-R) was measured through a 530/30-nm bandpass filter (Oriel Optical, Stamford, CT). The fixed cells were gated on the basis of forward-angle light-scatter pulse height, forward-angle light-scatter pulse width, and right-angle light-scatter pulse height to exclude small debris, cell fragments, and cell aggregates. Singleparameter data were collected for 10 000–20 000 intact viable cells by determination of forward
Fig. 1. Cell viability by trypan blue exclusion (top left) and FACS (bottom left) of Raji cells treated separately with: tumor promoters TPA and n-butyrate; smokeless tobacco purified products NNN, NNK and BaP; and smokeless tobacco dry snuff, moist snuff and loose leaf extracts (top left). Cell viability by trypan blue exclusion (top right) and FACS (bottom right) of Raji cells treated with TPA, NNN, NNK and BaP in combination with n-butyrate. Determinations of cell viability at each of the three time points (2, 4–5 and 6 – 7 days) were performed once for each of the smokeless tobacco purified products, three to six times for each extract (dry snuff, moist snuff, and loose leaf), five to eight times for TPA plus n-butyrate, and five to eight times for the untreated control cells. (The NNN plus n-butyrate sample for FACS analysis of viability at 6– 7 days was lost, although viability was determined by trypan blue staining.)
38 H.B. Jenson et al. / Toxicology 133 (1999) 35–42
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
39
Fig. 2. The viability of Raji cells determined using trypan blue (black columns) and using FACS (grey columns) after treatment with TPA plus n-butyrate. Induction of latent EBV was identified by EA-R expression using acetone () or paraformaldehyde () for cell fixation. The highest level of EA-R was seen at 6–7 days. The standard error is shown were multiple determinations were made. Cell viability was determined eight times each for days 2 and 4 – 5, and five times for day 6 – 7. EA-R after acetone fixation was determined three times for day 2, twice for day 4–5, and once for day 6 – 7 (and, therefore, the S.E. for this is undefined). EA-R after paraformaldehyde fixation was determined five times each for days 2 and 4 – 5, and four times for day 6 – 7.
and right-angle light-scatter for each experimental group, and the data were displayed as a cell frequency histogram over 512 channels. The percentage of cells that expressed EA-R (as determined by FACS analysis) was determined separately for each experimental group at the log relative fluorescence intensity at which 1% of the control cells stained with the primary antibody (mouse isotype-specific Ig2A monoclonal antibody) and the secondary antibody (goat antimouse IgG (heavy and light chain) – FITC conjugate antibody). For each experimental group, the percentage of EA-R detected by FACS in untreated Raji cells was subtracted to yield the percentage of Raji cells expressing EA-R resulting from the applied experimental treatment.
3. Results Determinations of cell viability at each of the
three time points (2, 4–5, and 6–7 days) were performed once for each of the smokeless tobacco purified products, three to six times for each extract (dry snuff, moist snuff, and loose leaf), five to eight times for TPA plus n-butyrate, and five to eight times for the untreated control cells. Cell viability determined by trypan blue exclusion by microscopy and by propidium iodide permeabilization by FACS showed similar findings (Fig. 1). The NNN plus n-butyrate sample for FACS analysis of viability at 6–7 days was lost, although viability was determined by trypan blue staining. TPA alone, n-butyrate alone, and TPA plus n-butyrate had the expected effects of cell death and a decreased percentage of viable cells. There was good reproducibility in the determination of viable cells in those cases where multiple determinations were made (Fig. 2). At the concentrations used, there was no significant toxic effect of
40
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
Table 1 Percent of Raji cells demonstrating Epstein–Barr virus induction, determined by FACS measurement of EA-R, after treatment with smokeless tobacco purified products and extractsa Treatment Paraformaldehyde fixation Untreated TPA n-butyrate TPA+n-butyrate NNN NNN+n-butyrate NNK NNK+n-butyrate BaP BaP+n-butyrate Dry snuff Moist snuff Loose leaf Acetone fixation Untreated TPA+n-butyrate Dry snuff Moist snuff Loose leaf
Day 2 (%9S.E.)
Day 4/5 (%9S.E.)
Day 6/7 (%9S.E.)
1.59 0.5b 0.6 0.4 4.89 1.2b 0 0 0 0.3 0.2 0.5 0.39 0.2d 0.4 9 0.2d 0.29 0.1d
1.0 90.2b 2.2 0.7 10.9 92.8b 0.6 0 0 0.6 0.4 0.4 0.05 90.05e 0.03 9 0.03d 0 90d
1.1 90.1c 3.9 0.5 9.9 96.2c 0.1 0.1 0.1 0.4 0 0.6 0.8 0.05 9 0.05e 0.1
1.69 0.3d 5.99 3.23d 0.39 0.05e 0.39 0.0e 0.6 9 0.6e
1.7 90.3e 3.2 92.1e 0 2.2 91.6e 0
1.6 15.2 0.4 0.3 0
a For each experimental group, the percentage of EA-R detected by FACS in untreated Raji cells was subtracted to yield the percentage of Raji cells expressing EA-R resulting from the applied experimental treatment. If more than one experiment was performed, the reported result is the mean 9 S.E. b Mean9 S.E. of five replicates. c Mean 9S.E. of four replicates. d Mean9 S.E. of three replicates. e Mean 9S.E. of two replicates.
smokeless tobacco purified products or extracts on Raji cells. Production of EA-R, indicative of activation of latent EBV in Raji cells, occurred with treatment with TPA alone, and most strongly with TPA plus n-butyrate at 6–7 days (Fig. 2). There was no consistent difference between acetone or paraformaldehyde fixation. The smokeless tobacco purified products or extracts did not have a significant effect on expression of EA-R when used individually, and did not have an additive effect when combined with n-butyrate (Table 1), as has been demonstrated by other known inducers of EBV (Ito et al., 1981). EA-R is stable after acetone fixation, unlike EA-D (Henle et al., 1970). Fixation of cells in paraformaldehyde has the potential advantage of permitting FACS analysis of both EA-R and EA-D
(Lidin et al., 1993). There was no difference in induction of latent EBV for any of the smokeless tobacco purified products or extracts, using either paraformaldehyde- or acetone-fixed cells (Table 1).
4. Discussion Smokeless tobacco contains numerous constituents that may act individually or synergistically with other environmental factors to promote the development of oral cancers (Hoffmann et al., 1994; Gupta et al., 1996; Hoffmann and Djordjevic, 1997). Smokeless tobacco-specific nitrosamines interact with Herpes simplex virus and demonstrate synergism for cell transformation, supporting a possible synergistic relationship for oral carcino-
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
genesis between Herpes simplex virus and smokeless tobacco (Park et al., 1991; Murrah et al., 1996). FACS detected a higher percentage of fluorescing (dead) cells than did the trypan blue exclusion method to determine cell viability. Both methods showed similar trends among and between treatments but, generally, lower percentage viabilities were detected using FACS (Table 1 and Fig. 2). FACS analysis of Raji cells for EA detection is more sensitive and more objective than immunofluorescence microscopy, and has the added advantage of rapid evaluation of much larger cell populations (20 000 cells or more); it may also be better suited for study of induction of latent EBV by weak inducing agents (Jenson et al., 1998). EBV is distributed worldwide. Although there are differences between different populations in the age-related incidence of primary EBV infection, EBV infects more than 98% of the world’s adult population. Primary EBV infection is followed by lifelong latent infection with intermittent oropharyngeal shedding and communicability. Approximately 12– 33% of healthy individuals shed EBV from the oropharynx at any given time, indicative of periodic active viral replication (Miller et al., 1973; Niederman et al., 1976). Immunocompromised persons, including persons with the acquired immunodeficiency syndrome (AIDS), shed EBV from the oropharynx at a significantly higher rate (49 – 78%) (Ferbas et al., 1992; Lucht et al., 1995). The other host factors and environmental factors that contribute to periodic reactivation of EBV are not well defined. Environmental factors, such as use of smokeless tobacco products, may contribute to increased risk of oral cancer by increasing EBV replication and associated inflammatory processes in the oral cavity. Our studies used experimental drug concentrations that have demonstrated carcinogenic effects of smokeless tobacco in other models (Murrah et al., 1993, 1996). In our studies, the smokeless tobacco purified products NNN, NNK and BaP, and smokeless tobacco extracts dry snuff, moist snuff and loose leaf, did not contribute to reactivation of EBV in vitro. There were no individual
41
effects and no additive effects when combined with n-butyrate on the induction of latent EBV.
References Boos, H., Berger, R., Kuklik-Roos, C., Iftner, T., MuellerLantzsch, N., 1987. Enhancement of Epstein – Barr virus membrane protein (LMP) expression by serum, TPA, or n-butyrate in latently infected Raji cells. Virology 159, 161 – 165. Bouvier, G., Poirier, S., Shao, Y.M., Malaveille, C., Ohshima, H., Polack, A., Bornkamm, G.W., Zeng, Y., de-The´, G., Bartsch, H., 1991. Epstein – Barr virus activators, mutagens and volatile nitrosamines in preserved food samples from high-risk areas for nasopharyngeal carcinoma. IARC Sci. Publ. 105, 204 – 209. Chang, R.S., Lewis, J.P., Abildgaard, C.F., 1973. Prevalence of oropharyngeal excreters of leukocyte-transforming agents among a human population. New Engl. J. Med. 289, 1325 – 1329. Chang, R.S., Lewis, J.P., Reynolds, R.D., Sullivan, M.J., Neuman, J., 1978. Oropharyngeal excretion of Epstein – Barr virus by patients with lymphoproliferative disorders and by recipients of renal homografts. Ann. Intern. Med. 88, 34 – 40. D’Costa, J., Saranath, D., Sanghvi, V., Mehta, A.R., 1998. Epstein – Barr virus in tobacco-induced oral cancers and oral lesions in patients from India. J. Oral Pathol. Med. 27, 78 – 82. Epstein, M.A., Achong, B.G., Barr, Y.M., Zajac, B., Henle, G., Henle, W., 1966. Morphological and virological investigations on cultured Burkitt tumor lymphoblasts (strain Raji). J. Natl. Cancer Inst. 37, 547 – 559. Ferbas, J., Rahman, M.A., Kingsley, L.A., Armstrong, J.A., Ho, M., Zhou, S.Y.J., Rinaldo, C.R. Jr., 1992. Frequent oropharyngeal shedding of Epstein – Barr virus in homosexual men during early HIV infection. AIDS 6, 1273 – 1278. Gerber, P., 1972. Activation of Epstein – Barr virus by 5-bromodeoxyuridine in ‘virus-free’ human cells. Proc. Natl. Acad. Sci. U.S.A. 69, 83 – 85. Gupta, P., Singh, M.K., Rinaldo, C., Ding, M., Farzadegan, H., Saah, A., Hoover, D., Moore, P., Kingsley, L., 1996. Detection of Kaposi’s sarcoma herpesvirus DNA in semen of homosexual men with Kaposi’s sarcoma. AIDS 10, 1596 – 1598. Hampar, B., Derge, J.G., Martos, L.M., Walker, J.L., 1971. Persistence of a repressed Epstein – Barr virus genome in Burkitt lymphoma cells made resistant to 5-bromodeoxyuridine. Proc. Natl. Acad. Sci. U.S.A. 68, 3185 – 3189. Hayashi, K., 1992. Effect of protein kinase C inhibitors with different action mechanisms on Epstein – Barr virus replication. Intervirology 33, 217 – 224.
42
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
Hecht, S.S., Rivenson, A., Braley, J., DiBello, J., Adams, J.D., Hoffmann, D., 1986. Induction of oral cavity tumors in F344 rats by tobacco-specific nitrosamines and snuff. Cancer Res. 46, 4162 – 4166. Henle, W., Henle, G., Zajac, B.A., Pearson, G., Waubke, R., Scriba, M., 1970. Differential reactivity of human serums with early antigens induced by Epstein–Barr virus. Science 169, 188 – 190. Hoffmann, D., Djordjevic, M.V., 1997. Chemical composition and carcinogenicity of smokeless tobacco. Adv. Dent. Res. 11, 322 – 329. Hoffmann, D., Rivenson, A., Chung, F.L., Hecht, S.S., 1991. Nicotine-derived N-nitrosamines (TSNA) and their relevance in tobacco carcinogenesis. Crit. Rev. Toxicol. 21, 305 – 311. Hoffmann, D., Brunnemann, K.D., Prokopczyk, B., Djordjevic, M.V., 1994. Tobacco-specific N-nitrosamines and Areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity, and relevance to humans. J. Toxicol. Environ. Health 41, 1 – 52. Ito, Y., Yanase, S., Fujita, J., Harayama, T., Takashima, M., Imanaka, H., 1981. A short-term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein–Barr virus. Cancer Lett. 13, 29– 37. Jenson, H.B., Grant, G.M., Ench, Y., Heard, P., Thomas, C.A., Hilsenbeck, S.G., Moyer, M.P., 1998. Immunofluorescence microscopy and flow cytometry characterization of chemical induction of latent Epstein–Barr virus. Clin. Diagn. Lab. Immunol. 5, 91–97. Kallin, B., Luka, J., Klein, G., 1979. Immunochemical characterization of Epstein –Barr virus-associated early and late antigens in n-butyrate-treated P3HR-1 cells. J. Virol. 32, 710 – 716. Lidin, B., Gartland, L., Marshall, G., Sanchez, V., Lamon, E.W., 1993. Detection of two early gene products of Epstein – Barr virus by fluorescence flow cytometry. Viral Immunol. 6, 97 – 107. Lucht, E., Biberfeld, P., Linde, A., 1995. Epstein–Barr virus (EBV) DNA in saliva and EBV serology of HIV-1-infected persons with and without hairy leukoplakia. J. Infect. 31, 189 – 194. Luka, J., Kallin, B., Klein, G., 1979. Induction of the Epstein – Barr virus (EBV) cycle in latently infected cells by n-butyrate. Virology 94, 228–231. Miller, G., Niederman, J.C., Andrews, L.-L., 1973. Prolonged oropharyngeal excretion of Epstein–Barr virus after infectious mononucleosis. New Engl. J. Med. 288, 229–232. Murphy, S.E., Heiblum, R., Trushin, N., 1990. Comparative metabolism of N%-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cultured F344 rat oral tissue and esophagus. Cancer Res. 50, 4685–4691. Murrah, V.A., Gilchrist, E.P., Moyer, M.P., 1993. Morphologic and growth effects of tobacco-associated chemical carcinogens and smokeless tobacco extracts on human oral
.
epithelial cells in culture. Oral Surg. Oral Med. Oral Pathol. 75, 323 – 332. Murrah, V.A., Gilchrist, E.P., Moyer, M.P., 1996. Attenuation of the natural course of herpes simplex virus infection in human oral epithelial cell cultures by smokeless tobacco extracts suggests the possibility of a synergistic mechanism for carcinogenesis. Oral Surg. Oral Med. Oral Pathol. Oral Rad. Endod. 81, 63 – 69. Niederman, J.C., Miller, G., Pearson, H.A., Pagano, J.S., Dowaliby, J.M., 1976. Infectious mononucleosis. Epstein – Barr-virus shedding in saliva and the oropharynx. New Engl. J. Med. 294, 1355 – 1359. Nonoyama, M., Pagano, J.S., 1972. Separation of Epstein – Barr virus DNA from large chromosomal DNA in nonvirus-producing cells. Nat. New Biol. 238, 169 – 171. Nutter, L.M., Grill, S.P., Li, J.S., Tan, R.S., Cheng, Y.C., 1987. Induction of virus enzymes by phorbol esters and n-butyrate in Epstein – Barr virus genome-carrying Raji cells. Cancer Res. 47, 4407 – 4412. Obi, F.O., Billett, M.A., 1991. Preferential binding of the carcinogen benzo[a]pyrene to proteins of the nuclear matrix. Carcinogenesis 12, 481 – 486. Ooka, T., Lenoir, G.M., Decaussin, G., Bornkamm, G.W., Daillie, J., 1986. Epstein – Barr virus-specific DNA polymerase in virus-nonproducer Raji cells. J. Virol. 58, 671 – 675. Park, N.H., Dokko, H., Li, S.L., Cherrick, H.M., 1991. Synergism of herpes simplex virus and tobacco-specific N%-nitrosamines in cell transformation. J. Oral Maxillofac. Surg. 49, 276 – 281. Pritchett, R., Pendersen, M., Kieff, E., 1976. Complexity of EBV homologous DNA in continuous lymphoblastoid cell lines. Virology 74, 227 – 231. Pulvertaft, R.J.V., 1965. A study of malignant tumours in Nigeria by short-term tissue culture. J. Clin. Pathol. 18, 261 – 273. Raab-Traub, N., 1996. Epstein – Barr virus: the virus and its tumorigenicity in the normal and immunocompromised host. Baillie`re’s Clin. Infect. Dis. 3, 457 – 475. Sairenji, T., Spiro, R.C., Humphreys, R.E., 1984. Differential effect of TPA and n-butyrate on induction of Ii and EBV antigens in the P3HR-1 lymphoblastoid cell line. Hematol. Oncol. 2, 381 – 389. Sciubba, J., Brandsma, J., Schwartz, M., Barrezueta, N., 1989. Hairy leukoplakia: an AIDS-associated opportunistic infection. Oral Surg. Oral Med. Oral Pathol. 67, 404 – 410. Tan, R.S., Li, J.S., Grill, S.P., Nutter, L.M., Cheng, Y.C., 1986. Demonstration of Epstein – Barr virus-specific DNA polymerase in chemically induced Raji cells and its antibody in serum from patients with nasopharyngeal carcinoma. Cancer Res. 46, 5024 – 5028. zur Hausen, H., O’Neill, F.J., Freese, U.K., Hecker, E., 1978. Persisting oncogenic herpesvirus induced by the tumour promotor TPA. Nature 272, 373 – 375.
Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein–Barr virus Hal B. Jenson a,b,*, Patty Heard a, Mary Pat Moyer b,c a
Department of Pediatrics, The Uni6ersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dri6e, San Antonio, TX 78284 -7811, USA b Department of Microbiology, The Uni6ersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dri6e, San Antonio, TX 78284 -7811, USA c Department of Surgery, The Uni6ersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dri6e, San Antonio, TX 78284 -7811, USA Received 23 September 1998; accepted 12 December 1998
Abstract Numerous chemical tumor promoters induce latent Epstein – Barr virus (EBV) to active replication. The effect of smokeless tobacco purified products N-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK), benzo(a)pyrene (BaP), and smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf tobacco) was tested for induction of latent EBV in Raji cells using fluorescence-activated cell sorter flow cytometry detection of the restricted component of EBV early antigen (EA-R). Concentrations of smokeless tobacco purified products or preparations were used that have carcinogenic effects in animal cell lines. There was no discernible effect for the 6–7-day duration of treatment on viability of Raji cells, or on induction of latent EBV in Raji cells. Results were comparable using paraformaldehyde- or acetone-fixed cells. There does not appear to be an in vitro effect of smokeless tobacco constituents on EBV-infected lymphocytes that may contribute to development of oral cancers. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Smokeless tobacco; Epstein–Barr virus; Virus induction; 12-O-tetradecanoyl phorbol-13-acetate; n-Butyrate; Fluorescence-activated cell sorter flow cytometry
1. Introduction
* Corresponding author. Present address: Department of Pediatrics, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7811, USA. Tel.: +1-210-5675301; fax: +1-210-5676305. E-mail address: [email protected] (H.B. Jenson)
Epstein–Barr virus (EBV) is associated with certain carcinomas, including nasopharyngeal carcinoma and Burkitt’s lymphoma (Raab-Traub, 1996), and is causally associated with oral hairy leukoplakia (Sciubba et al., 1989). EBV is shed intermittently from the oropharynx and can be
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 0 3 - 7
36
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
cultured from approximately 10 – 30% of immunocompetent persons (Chang et al., 1973) and from 50 – 100% of immunocompromised persons (Chang et al., 1978; Lucht et al., 1995). Chemicals known to induce replication of latent EBV infection in lymphocytes include: phorbol esters, including 12-O-tetradecanoyl phorbol-13acetate (TPA) (zur Hausen et al., 1978), a direct activator of protein kinase C (Hayashi, 1992); n-butyrate (Kallin et al., 1979; Luka et al., 1979); halogenated pyrimidines, including 5-iododeoxyuridine (Hampar et al., 1971; Gerber, 1972); and nitrosamines (Bouvier et al., 1991). Chemical induction by TPA (zur Hausen et al., 1978) leads to lytic EBV infection with a peak effect between 1 and 4 days (Sairenji et al., 1984; Boos et al., 1987; Nutter et al., 1987). The combination of EBV infection and tobacco may contribute to an increased incidence of oral cancers (D’Costa et al., 1998). Smokeless tobacco contains numerous chemicals with biologic activity and has been associated with numerous oral and pharyngeal cancers (Hoffmann et al., 1994; Gupta et al., 1996; Hoffmann and Djordjevic, 1997). The most abundant carcinogens in smokeless tobacco are the tobacco-specific nitrosamines, two of which, N-nitrosonornicotine (NNN) and 4 - (N - methyl - N - nitrosamine) - 1 - 3 - pyridinyl) - 1butanone (NNK), have been shown to increase longevity of mucosal cells in vitro with growth and morphologic changes suggestive of cell transformation (Murrah et al., 1993), and to induce benign and malignant tumors at various sites in mice, rats, and hamsters (Hecht et al., 1986; Hoffmann et al., 1991). Both NNN and NNK have been shown to pyridyloxobutylate DNA, and NNK has also been shown to methylate DNA (Murphy et al., 1990). Benzo(a)pyrene (BaP) is another carcinogenic compound found in smokeless tobacco (Obi and Billett, 1991). The Raji cell line, a human EBV genome-positive Burkitt’s lymphoma cell line, harbors approximately 50–60 EBV genome equivalents per cell (Nonoyama and Pagano, 1972; Pritchett et al., 1976). This non-producer cell line is particularly informative in the study of activation of latent EBV because of an intrinsic block after expression of the early antigens (EAs) that completely in-
hibits progression to viral DNA synthesis and late EBV gene expression (Ooka et al., 1986; Tan et al., 1986). The EA complex consists of several viral proteins expressed within the cell and is divided into two components, diffuse (EA-D) and restricted (EA-R), based on cellular localization (after fixation) and susceptibility to denaturation: EA-D is found in both the nucleus and cytoplasm, and is stable in acetone, methanol and ethanol; EA-R is found only in the cytoplasm, and is stable in acetone but denatured by methanol or ethanol (Henle et al., 1970). Latent EBV infection in the Raji cell line can be induced with TPA or n-butyrate, and can be measured by the determination of EBV early antigen (EA) production using fluorescence-activated cell sorter (FACS) flow cytometry (Jenson et al., 1998). The high prevalence of EBV infection, lifelong persistence after primary infection with replication in the oral cavity, and the carcinogens identified in smokeless tobacco and the association with oral cancers, suggest a possible cocarcinogenic interaction between EBV and smokeless tobacco. To determine the possible effect of smokeless tobacco on latent EBV, we measured virus induction in Raji cells after treatment with smokeless tobacco purified products (NNN, NNK, and BaP alone and in combination with n-butyrate (Ito et al., 1981) and tobacco extracts (dry snuff, moist snuff, and loose leaf), and compared these results to chemical induction by the tumor promoters TPA and n-butyrate, which are known to induce latent EBV.
2. Materials and methods
2.1. Smokeless tobacco extracts The tobacco extracts were prepared using standardized procedures (Murrah et al., 1993). Ten grams of either reference dry snuff, moist snuff, or loose leaf tobacco (Smokeless Tobacco Research Products, University of Kentucky, Lexington, KY) were briefly homogenized in 100 ml distilled, deionized H2O and extracted overnight at room temperature on an orbital shaker. After extrac-
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
tion, the solutions were centrifuged at 1800 × g for 30 min, and then centrifuged twice at 25000 × g for 30 min. The final supernatant was adjusted to pH 7.2–7.4 and sterile filtered through a 0.02mm syringe filter (Nalgene, Rochester, NY). The stock solution was designated as a 10% aqueous solution.
2.2. Raji cell line The Raji lymphoid cell line (ATCC CCL 86) is a human EBV-positive cell line derived from a Burkitt’s lymphoma (Pulvertaft, 1965; Epstein et al., 1966). The cells were maintained in RPMI 1640 medium (Cellgro, Mediatech, Herndon, VA) containing 10% fetal bovine serum (Hyclone, Logan, UT) with 100 U/ml of penicillin (Cellgro), 100 U/ml of streptomycin (Cellgro), 2.5 mg/ml of amphotericin B (Cellgro), and 2 mM glutamine (Cellgro) at 37°C in a humidified atmosphere with 5% CO2.
2.3. Chemical treatments Raji cells were suspended in fresh medium at 5 × 105 cells/ml and treated with one of the following chemicals: 1 mg/ml NNN (Midwest Research Institute, Kansas City, MO); 1 mg/ml NNK (Midwest Research Institute); 1 mg/ml BaP (Midwest Research Institute); 2% aqueous solution dry snuff; 2% aqueous solution moist snuff; 2% aqueous solution loose leaf tobacco (Smokeless Tobacco Research Products, University of Kentucky, Lexington, KY); 20 ng/ml TPA (Sigma, St. Louis, MO); or 4 mM n-butyric acid (Sigma). Combination treatments included NNN, NNK, BaP and TPA, each with n-butyrate (at the same concentrations used individually). The cells were harvested for analysis at 2, 4 – 5, and 6 – 7 days after treatment
2.4. Cell 6iability Absolute cell counts per milliliter and the proportion of viable cells were measured by trypan blue exclusion and by FACS flow cytometry at 2, 4 – 5 and 6–7 days after initiation of treatment. Cell viability was determined in separate aliquots
37
by trypan blue exclusion of 50 ml of cell suspension added to 0.4% trypan blue stain (GIBCO BRL, Grand Island, NY) at a 1:2 dilution. Dead cells were measured by permeabilization using propidium iodide fluorescence through a 630/22nm bandpass filter using a FACStar Plus (Becton Dickinson Immunocytometry Systems, San Jose, CA) flow cytometer with an argon-ion laser producing 200 mW of 488-nm light for excitation (Jenson et al., 1998)
2.5. Flow cytometry Cells were fixed and stained for EA using an adaptation of fixation that allows for flow cytometry analysis (Jenson et al., 1998). Cells were washed three times with phosphate buffered saline (PBS), fixed by resuspension in 1 ml of either acetone (EM Sciences, Gibbstown, NJ) or 2% paraformaldehyde (Sigma) with 0.1% Triton X (Sigma) at 4°C for 5 min (Lidin et al., 1993), and washed three times with a FACS buffer (PBS containing 10% goat serum albumin). EA-R was detected using mouse IgG2A monoclonal antibody against EA-R (Advanced Biotechnologies, Columbia, MD). The cells were stained with 20 ml of anti-EA-R antibody for 30 min at room temperature, rinsed three times with FACS buffer, and counterstained for 30 min at room temperature with goat anti-mouse IgG (heavy and light chain)–FITC conjugated antibody (Tago, Burlingame, CA) diluted 1:500 with 0.001% Evans blue in PBS. The cells were rinsed three times with FACS buffer and were resuspended in 0.5 ml of FACS buffer for FACS analysis. FACS flow cytometry was performed using a FACStar Plus (Becton Dickinson) flow cytometer with an argon-ion laser producing 200mW of 488-nm light for excitation. The fluorescence from the FITC (for EA-R) was measured through a 530/30-nm bandpass filter (Oriel Optical, Stamford, CT). The fixed cells were gated on the basis of forward-angle light-scatter pulse height, forward-angle light-scatter pulse width, and right-angle light-scatter pulse height to exclude small debris, cell fragments, and cell aggregates. Singleparameter data were collected for 10 000–20 000 intact viable cells by determination of forward
Fig. 1. Cell viability by trypan blue exclusion (top left) and FACS (bottom left) of Raji cells treated separately with: tumor promoters TPA and n-butyrate; smokeless tobacco purified products NNN, NNK and BaP; and smokeless tobacco dry snuff, moist snuff and loose leaf extracts (top left). Cell viability by trypan blue exclusion (top right) and FACS (bottom right) of Raji cells treated with TPA, NNN, NNK and BaP in combination with n-butyrate. Determinations of cell viability at each of the three time points (2, 4–5 and 6 – 7 days) were performed once for each of the smokeless tobacco purified products, three to six times for each extract (dry snuff, moist snuff, and loose leaf), five to eight times for TPA plus n-butyrate, and five to eight times for the untreated control cells. (The NNN plus n-butyrate sample for FACS analysis of viability at 6– 7 days was lost, although viability was determined by trypan blue staining.)
38 H.B. Jenson et al. / Toxicology 133 (1999) 35–42
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
39
Fig. 2. The viability of Raji cells determined using trypan blue (black columns) and using FACS (grey columns) after treatment with TPA plus n-butyrate. Induction of latent EBV was identified by EA-R expression using acetone () or paraformaldehyde () for cell fixation. The highest level of EA-R was seen at 6–7 days. The standard error is shown were multiple determinations were made. Cell viability was determined eight times each for days 2 and 4 – 5, and five times for day 6 – 7. EA-R after acetone fixation was determined three times for day 2, twice for day 4–5, and once for day 6 – 7 (and, therefore, the S.E. for this is undefined). EA-R after paraformaldehyde fixation was determined five times each for days 2 and 4 – 5, and four times for day 6 – 7.
and right-angle light-scatter for each experimental group, and the data were displayed as a cell frequency histogram over 512 channels. The percentage of cells that expressed EA-R (as determined by FACS analysis) was determined separately for each experimental group at the log relative fluorescence intensity at which 1% of the control cells stained with the primary antibody (mouse isotype-specific Ig2A monoclonal antibody) and the secondary antibody (goat antimouse IgG (heavy and light chain) – FITC conjugate antibody). For each experimental group, the percentage of EA-R detected by FACS in untreated Raji cells was subtracted to yield the percentage of Raji cells expressing EA-R resulting from the applied experimental treatment.
3. Results Determinations of cell viability at each of the
three time points (2, 4–5, and 6–7 days) were performed once for each of the smokeless tobacco purified products, three to six times for each extract (dry snuff, moist snuff, and loose leaf), five to eight times for TPA plus n-butyrate, and five to eight times for the untreated control cells. Cell viability determined by trypan blue exclusion by microscopy and by propidium iodide permeabilization by FACS showed similar findings (Fig. 1). The NNN plus n-butyrate sample for FACS analysis of viability at 6–7 days was lost, although viability was determined by trypan blue staining. TPA alone, n-butyrate alone, and TPA plus n-butyrate had the expected effects of cell death and a decreased percentage of viable cells. There was good reproducibility in the determination of viable cells in those cases where multiple determinations were made (Fig. 2). At the concentrations used, there was no significant toxic effect of
40
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
Table 1 Percent of Raji cells demonstrating Epstein–Barr virus induction, determined by FACS measurement of EA-R, after treatment with smokeless tobacco purified products and extractsa Treatment Paraformaldehyde fixation Untreated TPA n-butyrate TPA+n-butyrate NNN NNN+n-butyrate NNK NNK+n-butyrate BaP BaP+n-butyrate Dry snuff Moist snuff Loose leaf Acetone fixation Untreated TPA+n-butyrate Dry snuff Moist snuff Loose leaf
Day 2 (%9S.E.)
Day 4/5 (%9S.E.)
Day 6/7 (%9S.E.)
1.59 0.5b 0.6 0.4 4.89 1.2b 0 0 0 0.3 0.2 0.5 0.39 0.2d 0.4 9 0.2d 0.29 0.1d
1.0 90.2b 2.2 0.7 10.9 92.8b 0.6 0 0 0.6 0.4 0.4 0.05 90.05e 0.03 9 0.03d 0 90d
1.1 90.1c 3.9 0.5 9.9 96.2c 0.1 0.1 0.1 0.4 0 0.6 0.8 0.05 9 0.05e 0.1
1.69 0.3d 5.99 3.23d 0.39 0.05e 0.39 0.0e 0.6 9 0.6e
1.7 90.3e 3.2 92.1e 0 2.2 91.6e 0
1.6 15.2 0.4 0.3 0
a For each experimental group, the percentage of EA-R detected by FACS in untreated Raji cells was subtracted to yield the percentage of Raji cells expressing EA-R resulting from the applied experimental treatment. If more than one experiment was performed, the reported result is the mean 9 S.E. b Mean9 S.E. of five replicates. c Mean 9S.E. of four replicates. d Mean9 S.E. of three replicates. e Mean 9S.E. of two replicates.
smokeless tobacco purified products or extracts on Raji cells. Production of EA-R, indicative of activation of latent EBV in Raji cells, occurred with treatment with TPA alone, and most strongly with TPA plus n-butyrate at 6–7 days (Fig. 2). There was no consistent difference between acetone or paraformaldehyde fixation. The smokeless tobacco purified products or extracts did not have a significant effect on expression of EA-R when used individually, and did not have an additive effect when combined with n-butyrate (Table 1), as has been demonstrated by other known inducers of EBV (Ito et al., 1981). EA-R is stable after acetone fixation, unlike EA-D (Henle et al., 1970). Fixation of cells in paraformaldehyde has the potential advantage of permitting FACS analysis of both EA-R and EA-D
(Lidin et al., 1993). There was no difference in induction of latent EBV for any of the smokeless tobacco purified products or extracts, using either paraformaldehyde- or acetone-fixed cells (Table 1).
4. Discussion Smokeless tobacco contains numerous constituents that may act individually or synergistically with other environmental factors to promote the development of oral cancers (Hoffmann et al., 1994; Gupta et al., 1996; Hoffmann and Djordjevic, 1997). Smokeless tobacco-specific nitrosamines interact with Herpes simplex virus and demonstrate synergism for cell transformation, supporting a possible synergistic relationship for oral carcino-
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
genesis between Herpes simplex virus and smokeless tobacco (Park et al., 1991; Murrah et al., 1996). FACS detected a higher percentage of fluorescing (dead) cells than did the trypan blue exclusion method to determine cell viability. Both methods showed similar trends among and between treatments but, generally, lower percentage viabilities were detected using FACS (Table 1 and Fig. 2). FACS analysis of Raji cells for EA detection is more sensitive and more objective than immunofluorescence microscopy, and has the added advantage of rapid evaluation of much larger cell populations (20 000 cells or more); it may also be better suited for study of induction of latent EBV by weak inducing agents (Jenson et al., 1998). EBV is distributed worldwide. Although there are differences between different populations in the age-related incidence of primary EBV infection, EBV infects more than 98% of the world’s adult population. Primary EBV infection is followed by lifelong latent infection with intermittent oropharyngeal shedding and communicability. Approximately 12– 33% of healthy individuals shed EBV from the oropharynx at any given time, indicative of periodic active viral replication (Miller et al., 1973; Niederman et al., 1976). Immunocompromised persons, including persons with the acquired immunodeficiency syndrome (AIDS), shed EBV from the oropharynx at a significantly higher rate (49 – 78%) (Ferbas et al., 1992; Lucht et al., 1995). The other host factors and environmental factors that contribute to periodic reactivation of EBV are not well defined. Environmental factors, such as use of smokeless tobacco products, may contribute to increased risk of oral cancer by increasing EBV replication and associated inflammatory processes in the oral cavity. Our studies used experimental drug concentrations that have demonstrated carcinogenic effects of smokeless tobacco in other models (Murrah et al., 1993, 1996). In our studies, the smokeless tobacco purified products NNN, NNK and BaP, and smokeless tobacco extracts dry snuff, moist snuff and loose leaf, did not contribute to reactivation of EBV in vitro. There were no individual
41
effects and no additive effects when combined with n-butyrate on the induction of latent EBV.
References Boos, H., Berger, R., Kuklik-Roos, C., Iftner, T., MuellerLantzsch, N., 1987. Enhancement of Epstein – Barr virus membrane protein (LMP) expression by serum, TPA, or n-butyrate in latently infected Raji cells. Virology 159, 161 – 165. Bouvier, G., Poirier, S., Shao, Y.M., Malaveille, C., Ohshima, H., Polack, A., Bornkamm, G.W., Zeng, Y., de-The´, G., Bartsch, H., 1991. Epstein – Barr virus activators, mutagens and volatile nitrosamines in preserved food samples from high-risk areas for nasopharyngeal carcinoma. IARC Sci. Publ. 105, 204 – 209. Chang, R.S., Lewis, J.P., Abildgaard, C.F., 1973. Prevalence of oropharyngeal excreters of leukocyte-transforming agents among a human population. New Engl. J. Med. 289, 1325 – 1329. Chang, R.S., Lewis, J.P., Reynolds, R.D., Sullivan, M.J., Neuman, J., 1978. Oropharyngeal excretion of Epstein – Barr virus by patients with lymphoproliferative disorders and by recipients of renal homografts. Ann. Intern. Med. 88, 34 – 40. D’Costa, J., Saranath, D., Sanghvi, V., Mehta, A.R., 1998. Epstein – Barr virus in tobacco-induced oral cancers and oral lesions in patients from India. J. Oral Pathol. Med. 27, 78 – 82. Epstein, M.A., Achong, B.G., Barr, Y.M., Zajac, B., Henle, G., Henle, W., 1966. Morphological and virological investigations on cultured Burkitt tumor lymphoblasts (strain Raji). J. Natl. Cancer Inst. 37, 547 – 559. Ferbas, J., Rahman, M.A., Kingsley, L.A., Armstrong, J.A., Ho, M., Zhou, S.Y.J., Rinaldo, C.R. Jr., 1992. Frequent oropharyngeal shedding of Epstein – Barr virus in homosexual men during early HIV infection. AIDS 6, 1273 – 1278. Gerber, P., 1972. Activation of Epstein – Barr virus by 5-bromodeoxyuridine in ‘virus-free’ human cells. Proc. Natl. Acad. Sci. U.S.A. 69, 83 – 85. Gupta, P., Singh, M.K., Rinaldo, C., Ding, M., Farzadegan, H., Saah, A., Hoover, D., Moore, P., Kingsley, L., 1996. Detection of Kaposi’s sarcoma herpesvirus DNA in semen of homosexual men with Kaposi’s sarcoma. AIDS 10, 1596 – 1598. Hampar, B., Derge, J.G., Martos, L.M., Walker, J.L., 1971. Persistence of a repressed Epstein – Barr virus genome in Burkitt lymphoma cells made resistant to 5-bromodeoxyuridine. Proc. Natl. Acad. Sci. U.S.A. 68, 3185 – 3189. Hayashi, K., 1992. Effect of protein kinase C inhibitors with different action mechanisms on Epstein – Barr virus replication. Intervirology 33, 217 – 224.
42
H.B. Jenson et al. / Toxicology 133 (1999) 35–42
Hecht, S.S., Rivenson, A., Braley, J., DiBello, J., Adams, J.D., Hoffmann, D., 1986. Induction of oral cavity tumors in F344 rats by tobacco-specific nitrosamines and snuff. Cancer Res. 46, 4162 – 4166. Henle, W., Henle, G., Zajac, B.A., Pearson, G., Waubke, R., Scriba, M., 1970. Differential reactivity of human serums with early antigens induced by Epstein–Barr virus. Science 169, 188 – 190. Hoffmann, D., Djordjevic, M.V., 1997. Chemical composition and carcinogenicity of smokeless tobacco. Adv. Dent. Res. 11, 322 – 329. Hoffmann, D., Rivenson, A., Chung, F.L., Hecht, S.S., 1991. Nicotine-derived N-nitrosamines (TSNA) and their relevance in tobacco carcinogenesis. Crit. Rev. Toxicol. 21, 305 – 311. Hoffmann, D., Brunnemann, K.D., Prokopczyk, B., Djordjevic, M.V., 1994. Tobacco-specific N-nitrosamines and Areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity, and relevance to humans. J. Toxicol. Environ. Health 41, 1 – 52. Ito, Y., Yanase, S., Fujita, J., Harayama, T., Takashima, M., Imanaka, H., 1981. A short-term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein–Barr virus. Cancer Lett. 13, 29– 37. Jenson, H.B., Grant, G.M., Ench, Y., Heard, P., Thomas, C.A., Hilsenbeck, S.G., Moyer, M.P., 1998. Immunofluorescence microscopy and flow cytometry characterization of chemical induction of latent Epstein–Barr virus. Clin. Diagn. Lab. Immunol. 5, 91–97. Kallin, B., Luka, J., Klein, G., 1979. Immunochemical characterization of Epstein –Barr virus-associated early and late antigens in n-butyrate-treated P3HR-1 cells. J. Virol. 32, 710 – 716. Lidin, B., Gartland, L., Marshall, G., Sanchez, V., Lamon, E.W., 1993. Detection of two early gene products of Epstein – Barr virus by fluorescence flow cytometry. Viral Immunol. 6, 97 – 107. Lucht, E., Biberfeld, P., Linde, A., 1995. Epstein–Barr virus (EBV) DNA in saliva and EBV serology of HIV-1-infected persons with and without hairy leukoplakia. J. Infect. 31, 189 – 194. Luka, J., Kallin, B., Klein, G., 1979. Induction of the Epstein – Barr virus (EBV) cycle in latently infected cells by n-butyrate. Virology 94, 228–231. Miller, G., Niederman, J.C., Andrews, L.-L., 1973. Prolonged oropharyngeal excretion of Epstein–Barr virus after infectious mononucleosis. New Engl. J. Med. 288, 229–232. Murphy, S.E., Heiblum, R., Trushin, N., 1990. Comparative metabolism of N%-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cultured F344 rat oral tissue and esophagus. Cancer Res. 50, 4685–4691. Murrah, V.A., Gilchrist, E.P., Moyer, M.P., 1993. Morphologic and growth effects of tobacco-associated chemical carcinogens and smokeless tobacco extracts on human oral
.
epithelial cells in culture. Oral Surg. Oral Med. Oral Pathol. 75, 323 – 332. Murrah, V.A., Gilchrist, E.P., Moyer, M.P., 1996. Attenuation of the natural course of herpes simplex virus infection in human oral epithelial cell cultures by smokeless tobacco extracts suggests the possibility of a synergistic mechanism for carcinogenesis. Oral Surg. Oral Med. Oral Pathol. Oral Rad. Endod. 81, 63 – 69. Niederman, J.C., Miller, G., Pearson, H.A., Pagano, J.S., Dowaliby, J.M., 1976. Infectious mononucleosis. Epstein – Barr-virus shedding in saliva and the oropharynx. New Engl. J. Med. 294, 1355 – 1359. Nonoyama, M., Pagano, J.S., 1972. Separation of Epstein – Barr virus DNA from large chromosomal DNA in nonvirus-producing cells. Nat. New Biol. 238, 169 – 171. Nutter, L.M., Grill, S.P., Li, J.S., Tan, R.S., Cheng, Y.C., 1987. Induction of virus enzymes by phorbol esters and n-butyrate in Epstein – Barr virus genome-carrying Raji cells. Cancer Res. 47, 4407 – 4412. Obi, F.O., Billett, M.A., 1991. Preferential binding of the carcinogen benzo[a]pyrene to proteins of the nuclear matrix. Carcinogenesis 12, 481 – 486. Ooka, T., Lenoir, G.M., Decaussin, G., Bornkamm, G.W., Daillie, J., 1986. Epstein – Barr virus-specific DNA polymerase in virus-nonproducer Raji cells. J. Virol. 58, 671 – 675. Park, N.H., Dokko, H., Li, S.L., Cherrick, H.M., 1991. Synergism of herpes simplex virus and tobacco-specific N%-nitrosamines in cell transformation. J. Oral Maxillofac. Surg. 49, 276 – 281. Pritchett, R., Pendersen, M., Kieff, E., 1976. Complexity of EBV homologous DNA in continuous lymphoblastoid cell lines. Virology 74, 227 – 231. Pulvertaft, R.J.V., 1965. A study of malignant tumours in Nigeria by short-term tissue culture. J. Clin. Pathol. 18, 261 – 273. Raab-Traub, N., 1996. Epstein – Barr virus: the virus and its tumorigenicity in the normal and immunocompromised host. Baillie`re’s Clin. Infect. Dis. 3, 457 – 475. Sairenji, T., Spiro, R.C., Humphreys, R.E., 1984. Differential effect of TPA and n-butyrate on induction of Ii and EBV antigens in the P3HR-1 lymphoblastoid cell line. Hematol. Oncol. 2, 381 – 389. Sciubba, J., Brandsma, J., Schwartz, M., Barrezueta, N., 1989. Hairy leukoplakia: an AIDS-associated opportunistic infection. Oral Surg. Oral Med. Oral Pathol. 67, 404 – 410. Tan, R.S., Li, J.S., Grill, S.P., Nutter, L.M., Cheng, Y.C., 1986. Demonstration of Epstein – Barr virus-specific DNA polymerase in chemically induced Raji cells and its antibody in serum from patients with nasopharyngeal carcinoma. Cancer Res. 46, 5024 – 5028. zur Hausen, H., O’Neill, F.J., Freese, U.K., Hecker, E., 1978. Persisting oncogenic herpesvirus induced by the tumour promotor TPA. Nature 272, 373 – 375.