Teas and tea components as inhibitors of carcinogen formation in model systems and man.

BACKGROUND: Epidemiological evidence points to a cancer protective role of green-yellow-orange vegetables and fruits. The involvement of teas as a protective factor in carcinogenesis has not received the attention it seems to merit. To gain relevant information, attempts were made to stimulate in vitro those conditions to which human groups are actually exposed. METHODS: The inhibitory effects of infusions of Chinese, Japanese, and Ceylonese teas were examined by adding them to a nitrosation mixture consisting of 0.8 mg sodium nitrite and 340 mg equivalent of a widely consumed salt-preserved fish (Pak Wik) and estimating the frequency of mutants in TA 1535 strain of Salmonella typhimurium. RESULTS: The tea samples exhibited a strong inhibitory effect at concentrations that are actually ingested by man. A comparable inhibition was obtained by several tea phenolics. A second series of experiments dealt with the formation of nitrosoproline (NPRO) which can be strongly inhibited in vitro by the tea infusions and tea phenolics. The effects of the tea infusions and caffeic acid on the endogeneous formation of NPRO in man were examined by having volunteers ingest 300 mg sodium nitrate and 30 min later 300 mg proline, collecting urine samples over a 24-hr period, and estimating the excreted NPRO. The tested teas, at doses regularly consumed, again exerted a strong inhibitory effect on endogeneous NPRO formation in humans. Comparable inhibitory effects were obtained by ingesting caffeic acid, chlorogenic acid, or ferulic acid with the nitrosation mixture. CONCLUSIONS: These results indicate that the simultaneous intake of teas with food products that are being nitrosated within the stomach of human subjects should exert a protective, beneficial effect.

Clastogenic and mutagenic actions of active species generated in the 6-hydroxydopamine/oxygen reaction: effects of scavengers of active oxygen, iron, and metal chelating agents.

A pro-oxidant triphenol, 6-hydroxydopamine (6-OHDA), induced mutations in the Salmonella typhimurium TA 104 tester strain (over the concentration range to 800 microM), and induced chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells at lower concentrations (up to 90 microM). It was however only marginally mutagenic (up to cytotoxic levels of 200 microM) in the TA102 tester strain. Clastogenicity in the more sensitive CHO cell assay was mediated by activated oxygen. Superoxide dismutase decreased the incidence of chromosomal aberrations by 60% and catalase (or superoxide dismutase plus catalase) decreased the incidence to control levels. The clastogenicity of 6-OHDA was dependent upon unsequestered transition metal ions, since addition of EDTA plus desferrioxamine decreased chromosomal aberrations by 90%. The simplest explanation of the data is that genotoxicity is mediated by active species generated in a Fenton-type reaction between 6-OHDA and H2O2 catalyzed by traces of metals in the medium.

Localized formation of micronuclei in the oral mucosa and tobacco-specific nitrosamines in the saliva of "reverse" smokers, Khaini-tobacco chewers and gudakhu users.

"Reverse"-cigar smokers (who hold the burning end of cigars within the mouth), dippers (who place a mixture of Khaini-tobacco and slaked lime into the lower gingival groove) and users of tobacco-containing toothpaste (gudakhu) in Orissa, India, were examined for precancerous oral lesions, the frequency of micronucleated cells at 3 different intra-oral sites, and levels of tobacco-specific nitrosamines (TSNA) in the saliva. Among reverse-cigar smokers, a high incidence of leukokeratosis nicotina palati, an elevated frequency of micronucleated cells in the palate (2.5% as compared to 0.6% in non-smokers and non-chewers of tobacco) and tongue (2.1%) from which carcinomas preferentially develop, and up to 5890 ppb nitrosonornicotine and up to 1880 ppb N-nitrosoanatabine in the saliva were found. Among Khaini-tobacco chewers, the frequency of micronucleated cells was elevated to 2.1% in the gingival groove, and up to 1580 ng N-nitrosonornicotine, 690 ng N-nitrosoanatabine, 90 ng N-nitrosoanabasine, and 180 ng 4-(methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone) per ml of saliva were observed. The localized elevation of the frequency of micronuclei and cancer development is probably due to a synergistic effect of hyperthermia and tobacco-related carcinogens among reverse-cigar smokers, and to the close, prolonged contact between the mucosa and tobacco among Khaini-tobacco/slaked lime dippers. Neither pre-cancerous lesions nor an elevated frequency of micronuclei were seen in the oral mucosa of users of gudakhu, a tobacco-containing toothpaste, which may be due to the low amount of TSNA released from the gudakhu and the short exposure time, which is restricted to the period of tooth brushing.

DNA adducts in bronchial biopsies.

To investigate the feasibility of measuring DNA-carcinogen adducts in the lungs of non-surgical patients, endobronchial biopsies were obtained from 78 patients undergoing routine diagnostic bronchoscopy. Lung cancer was present in 37 (47%) of the patients. DNA was isolated from the tissues and analyzed by HPLC- or nuclease-PI-enriched 32P-postlabelling, using procedures selective for aromatic adducts. Chromatograms from all 28 current smokers showed a distinctive diagonal adduct zone which was present in only 24 of 40 ex-smokers and 4 of 10 lifetime non-smokers. Adduct levels and chromatographic patterns were similar in bronchial tissue from different lobes of the lung, in bronchial and alveolar tissue, and in tumor and non-tumor bronchial tissue taken from the same subject. Bronchial DNA adduct levels were strongly associated with cigarette smoking status and dropped rapidly after smoking ceased. Higher levels of DNA adducts seen in the lung-cancer patients were mainly due to cigarette smoking. Frequent alcohol intake was the only dietary factor associated with higher levels of bronchial DNA adducts. We conclude that the level of bronchial DNA adducts is strongly associated with cigarette-smoking history and with alcohol intake, but is not associated with lung cancer independently from its relation to smoking. The results indicate the feasibility of using 32P-postlabelling to detect and quantitate genetic damage in bronchial biopsy specimens.

The beneficial and hazardous effects of simple phenolic compounds.

The current emphasis on screening the environment for man-made genotoxic and carcinogenic compounds detracts from studies on the possible health hazard or beneficial effects of naturally occurring agents to which humans are exposed daily. The simple phenolics, which are ubiquitous among plants, used as food additives, and ingested daily in milligram quantities, belong to this category of compounds. They induce double-strand DNA breaks. DNA adducts, mutations and chromosome aberrations in a great variety of test systems. However, they can suppress the genotoxic activity of numerous carcinogenic compounds in both in vitro and in vivo assays. This dual function of dietary phenolics also becomes evident when their carcinogenic or anticarcinogenic potential is examined. Some, but not all, phenolics induce precancerous lesions, papillomas and cancers, act as cocarcinogens, and exert a promoting effect in various rodent assays. On the other hand, phenolics have proved to be potent inhibitors of carcinogenesis at the initiation and promotion stages induced by carcinogens and promoters of different molecular structures. The extent to which a health hazard or protective activity of complex dietary mixtures is due to their phenolic content remains an unresolved issue. In addition, these multiple, occasionally contradictory functions of simple phenolics make it difficult to propose their use as chemopreventive agents.

Remission of precancerous lesions in the oral cavity of tobacco chewers and maintenance of the protective effect of beta-carotene or vitamin A.

Participants in the intervention trials were fishermen (Kerala, India), who chewed tobacco-containing betel quids daily before and throughout the study period. Frequency of oral leukoplakia, micronuclei in oral mucosal cells, and alterations in nuclear textures were used as endpoints. Administration of vitamin A (60 mg/wk) for 6-mo resulted in complete remission of leukoplakias in 57% and a reduction of micronucleated cells in 96% of tobacco-chewers. beta-carotene (2.2 mmol/wk) induced remission of leukoplakia in 14.8% and reduction of micronucleated cells in 98%. Vitamin A completely suppressed and beta-carotene suppressed by 50% formation of new leukoplakia within the 6-mo trial period. After withdrawal of vitamin A or beta-carotene treatment, oral leukoplakias reappeared, frequency of micronuclei in oral mucosa increased, and nuclear textures reverted to those present before the administration of chemo-preventive agents. The protective effect of the original treatment could be maintained for at least 8 additional months by administration of lower doses of vitamin A or beta-carotene.

Remission of oral precancerous lesions of tobacco/areca nut chewers following administration of beta-carotene or vitamin A, and maintenance of the protective effect.

Designs of intervention trials are based on results from a multitude of disciplines. In this study, a comparative analysis of various chewing mixtures used by groups from different geographical locations (Guam, Peru, Taiwan, the Philippines, and India) and their link to oral cancer incidences was used to trace the ingredients responsible for oral carcinogenesis among chewers. The usefulness of applying intermediate endpoints in intervention trials was examined by comparing the response of micronucleated mucosal cells and oral leukoplakia of chewers of tobacco-containing betel quids to the twice weekly administration of beta-carotene (180 mg/week), vitamin A (100,000 IU/week or 200,000 IU/week), and beta-carotene (180 mg/week) plus vitamin A (100,000 IU/week). A reduced frequency of micronucleated mucosal cells and remission of leukoplakias resulted following a 3- to 6-month treatment. The development of new leukoplakias was also inhibited. The various endpoints differed in degree and time course to the administration of beta-carotene and vitamin A. Following termination of the beta-carotene or vitamin A administration, micronucleated cells and leukoplakia recurred in the oral cavity of chewers who continued this habit throughout the trial period. Attempts were made to maintain the protective effect achieved by the treatment with relatively high doses of the chemopreventive agents. Vitamin A given at a level of 50,000 IU/week was able to keep the frequency of micronucleated mucosal cells at low levels for at least a 12-month post-treatment period, whereas beta-carotene administered at 60 mg/week was less effective in maintaining the protective effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell transformation induced by bovine papillomavirus DNA as an assay for tumor promoters and chemopreventive agents.

An in vitro assay was designed to examine and quantitate the action of chemical promoters and chemopreventive agents on papillomavirus DNA-carrying cells. Cultured C3H/10T1/2 cells transfected with bovine papillomavirus type 1 DNA (plasmid pdBPV-1) were used as targets, and the frequency of transformed foci was used as an endpoint. The development of foci with a transformed phenotype was greatly enhanced by tumor promoters (e.g., mezerein, 12-O-tetradecanoyl-phorbol-13-acetate, teleocidin, and okadaic acid) and complex mixtures such as extracts of the areca nut, which is an integral part of a betel quid and is linked to oral cancers among chewers. The degree of promotion depended on the length of exposure, the type of promoter, and the time of application after transfection with BPV DNA. The inhibitory effect of chemopreventive agents on transformation can be tested either directly on BPV DNA transfected cells (promoter-independent transformation), or on transfected cells that were exposed to tumor promoters (promoter-dependent transformation). Retinol, and to a lesser degree beta-carotene, exerted an inhibitory effect on promoter-dependent and promoter-independent transformation of BPV DNA transfected cells. The inhibitory effect was conveyed either by the addition of retinol simultaneously with promoters, or after exposure to the promoting agents was completed. The significance of this short-term in vitro assay for the design of chemopreventive trials is discussed.

The effect of retinoids, carotenoids and phenolics on chromosomal instability of bovine papillomavirus DNA-carrying cells.

Antioxidants were found to protect against the genotoxic effects of chemical and physical mutagenic and clastogenic agents. This study focused on the capacity of antioxidants to reduce an intrinsic and persistent chromosome instability. As a model system, strains of C127 cells, which were transformed by bovine papillomavirus (BPV) DNA and which carry BPV DNA varying from 20 to 160 copies, were used. Transformed cells of 10 different strains showed a persistently high incidence of mitotic irregularities detectable at anaphase and telophase (27.3-58.9%), an elevated frequency of cells with micronuclei (6.6-34.7%), and a broad spectrum of nuclear sizes, as measured by image analysis. A 3-day exposure to retinoic acid, retinol, beta-carotene, canthaxanthin, ascorbic acid and ellagic acid greatly reduced the degree of chromosome instability, whereas catechin, eugenol and pyrogallol showed a smaller inhibitory effect, and curcumin had no detectable effect on the frequency of mitotic irregularities. After withdrawal of retinoic acid treatment, the high levels of chromosome instability reappeared. The possibility that the protective effect of the retinoids and carotenoids examined in the model system points to their beneficial administration to human cells with an intrinsic or acquired chromosome instability is discussed.

Variations in the uptake of beta-carotene in the oral mucosa of individuals after 3 days of supplementation.

Variations in the response of individuals to beta-carotene supplementation were studied by measuring the accumulation of beta-carotene in oral mucosa cells. Beta-carotene was administered orally to 178 individuals for 3 consecutive days, exfoliated oral mucosa cells were collected by brushing the entire oral mucosa on the 7th day following supplementation, and the beta-carotene content was measured by HPLC analysis of the pronase-treated cells. The rise in beta-carotene levels in the oral mucosa following supplementation varied considerably. Significant differences in mean beta-carotene levels in the oral mucosa were observed in 4 population groups. After supplementation, beta-carotene levels increased by factors of 10.3 (100 Mile House), 7.8 (Williams Lake), 6.9 (Lytton) and 3.4 (Vancouver), respectively. This difference in mean beta-carotene values is due to there being different proportions of weak and strong responders in the various population groups. Neither peak levels nor increases in beta-carotene levels were correlated with base-line concentrations of beta-carotene in the pre-supplementation samples. A second supplementation was given to 54 individuals several months after the initial supplementation. Of 17 weak responders in the first supplementation study, 10 (58.8%) individuals again showed only a small increase. A time-course study revealed that low responders showed no significant changes in beta-carotene levels over the 21 days following supplementation. Particular attention should be paid to weak responders when results of intervention trials using beta-carotene are interpreted.

Quantitation of chromatin patterns by image analysis as a predictive tool in chemopreventive trials with vitamin A.

Nuclear textures of oral mucosal cells were quantitated by image analysis, and their suitability as markers in a chemopreventive trial explored. Subjects were chewers of tobacco-containing betel quids with well established oral leukoplakias. Treatment consisted of a weekly oral administration of vitamin A (200,000 IU/week) for six months. Leukoplakias regressed in 57.1% of the 21 trial participants. The original leukoplakias did not redevelop within four months after termination of treatment. For image analysis, biopsies were taken from leukoplakias of five chewers before administration of vitamin A, at the end of the administration, and four months after termination of treatment. Sections of paraffin-embedded biopsies were stained with the Feulgen reaction and submitted to quantitative image analysis of two parameters: variance of intensity and entropy. Both these parameters were significantly reduced in all five trial participants as a result of the six-month vitamin A treatment. During the post-treatment period, nuclei with condensed chromatin, as measured by the variance of intensity, reappeared in the mucosa of four of the five chewers examined, although no leukoplakias were detectable on visual examination of the oral cavity. The results indicate that the quantitation of nuclear textures in a small subpopulation of a chemopreventive trial could conceivably be a simple marker with a predictive value.

The involvement of reactive oxygen species in oral cancers of betel quid/tobacco chewers.

Most biological reactions, including carcinogenesis, are complex processes involving thousands of compounds, their metabolites and intermediates. The separation of events which form part of a direct chain leading to neoplastic transformation from those which are mere by-products is a herculean task. In this study, we focused on the pros and cons of reactive oxygen species (ROS) being involved in the development of oral cancer among chewers of tobacco and areca nuts. The results revealed that bursts of ROS generation occur at different stages of carcinogenesis, and are caused by different mechanisms. This observation may have considerable practical implications. Different strategies will be required in the administration of chemopreventive agents in order to trap ROS formed in the alkaline (due to the addition of slaked lime) chewing mixture within the saliva of a chewer, to scavenge ROS within mucosal cells exposed to an array of tobacco- or areca nut-related carcinogens or tumour promoters, and to inhibit the action of ROS released from ROS-generating white cells during lymphocytic infiltration of the oral mucosa at a precancerous stage. The remission of oral leukoplakias following the administration of vitamin A (200,000 IU/week) or vitamin A (100,000 IU/week) plus beta-carotene (180 mg/week) for 6 months, the inhibition of new leukoplakias during this trial period, and the reduction of micronucleated oral mucosal cells in chewers treated with beta-carotene or vitamin A are indeed promising results. However, a better understanding of the role of ROS in various stages of carcinogenesis will provide the basis for selection of the proper chemopreventive agents and the design of a treatment regime which may either prevent the formation of precancerous lesions, induce their remission, or inhibit the progression of precancerous lesions into malignant cancers.

Chemopreventive trials with vitamin A and beta-carotene: some unresolved issues.

The administration of beta-carotene (180 mg/week) plus vitamin A (100,000 IU/week) or vitamin A alone (200,000 IU/week) to chewers of betel quids in Kerala, India led to a reduction in the frequency of micronucleated buccal mucosal cells, a remission of oral leukoplakia, and an inhibition of the development of new leukoplakias. The advantages of this test system include a profound knowledge of exposure levels to tobacco-specific nitrosamines, areca nut-specific nitrosamines, and reactive oxygen species (ROS)-generating polyphenolics; the ease of quantitating micronuclei in exfoliated buccal mucosal cells and oral leukoplakia by noninvasive procedures; and solid information on the incidence of preneoplastic lesions and carcinomas. Some practical issues such as the level of nontoxic beta-carotene which could maintain the reduced frequency of micronucleated cells and the remission of oral leukoplakias for prolonged periods of time, the logistics of distributing beta-carotene to the many millions of smokeless tobacco users who are at elevated risk for oral cancer, and the possibility of using beta-carotene in the form of sweet potatoes or red palm oil, which could contain an economical source of beta-carotene for developing countries in which most of the tobacco chewers live, still remain unresolved.

Use of exfoliated cells to study tissue-specific levels of beta-carotene in humans.

This study was designed to explore the feasibility of using exfoliated cells to study beta-carotene incorporation into different epithelial tissues in humans. Exfoliated cells were collected from the oral cavities (by brushing the oral mucosa) and from the urogenital tracts (by centrifuging urine samples) of 36 females and basal levels of beta-carotene (without oral supplementation) were determined. Beta-carotene levels in cells from the two sites differed significantly, although a weak correlation was observed. As a second aspect of the study, 10 of these females were given oral supplementation with beta-carotene (90 mg twice weekly for 4 weeks). Beta-carotene levels increased significantly in both exfoliated urogenital tract (6.8-fold) and oral mucosa (5-fold) cells. However, the supplemented levels remained significantly different for the two types of cells. Beta-carotene levels did not change in individuals receiving a placebo treatment (n = 7). These studies suggest that exfoliated cells collected from different sites may be of value in quantifying tissue levels of beta-carotene during cancer intervention trials.

Promoting activity of betel quid ingredients and their inhibition by retinol.

Ingredients of betel quids, which have been linked to the high incidence of precancerous oral lesions and oral cancers, were examined for their promoting activity. Aqueous extracts were tested using the bovine papillomavirus (BPV) DNA transformation assay, which consists of cultured C3H/10T1/2 cells transfected with the plasmid pdPBV-1 as targets, and the frequency of transformed foci as endpoints. Areca nut extracts enhanced the formation of BPV DNA-induced transformed foci approximately tenfold. No promoting activity was detected in two samples of chewing tobacco examined. The addition of retinol to the areca nut extract inhibited its tumour promoting effect in a dose-dependent manner, completely abolishing the promoting activity at a dose of 10(-6) M. The experimental results are compared with epidemiological data on oral cancer incidences among chewers of different areca nut/tobacco mixtures and with the chemopreventive effect of vitamin A administered to betel quid chewers.

Changes in DNA copy numbers of bovine papillomavirus type 1 after termination of retinoic acid treatment.

The number of bovine papillomavirus type 1 (BPV) DNA copies [plasmid pdBPV-1 (142-6)] was examined in transformed C127 cells of an RIII mouse during exposure to all-trans-retinoic acid (RA) and after its withdrawal. RA treatment of a transformed cell line reduced the number from approximately 60 copies to an average of less than one copy per cell within 5 weeks. The composition of the RA-treated cell population was heterogeneous with respect to BPV DNA copies: 89.7% of the cells had no detectable copies, 8.6% had one copy, 1.7% had fewer than five copies, and one in 13,000 cells carried more than 10 copies. The low number of BPV DNA copies in the RA-treated cell population did not increase when the cells were subcultured before reaching confluence. RA-treated cell populations that contained less than one BPV DNA copy lost the transformed phenotype. However, a small fraction of cells (1 in 13,000) with greater than or equal to 10 BPV DNA copies retained the capacity to develop into transformed colonies. The relevance of these results to the regression of papillomavirus, DNA-carrying human lesions after exposure to retinoids and the redevelopment of these lesions after withdrawal of retinoids is discussed.

Enhancement of bovine papillomavirus-induced cell transformation by tumour promoters.

Cultured C3H/10T1/2 cells transfected with the plasmid pdBPV-1 were used as targets, and the frequency of transformed colonies as the endpoint to test the enhancing capacity of four promoters: 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA), mezerein and phorbol-12-retinoate-13-acetate (PRA). The frequency of the transfected C3H/10T1/2 cells to form transformed colonies was enhanced in the following order: mezerein greater than PRA greater than TPA greater than 4-O-methyl-TPA. The amount of promoters required to promote a tenfold increase in transformed cells was 0.24, 0.81, 30 and 100 ng/ml mezerein, PRA, TPA and 4-O-methyl-TPA, respectively. A significant promoting effect was obtained by a 3.5-day exposure to mezerein regardless of whether it was added at different time intervals after transfection with BPV-DNA. The examined promoters lacked genotoxic activity, as tested on Chinese hamster ovary cells, using chromatid aberrations and exchanges, frequency of macronuclei, unscheduled DNA synthesis (UDS) and inhibition of UDS as endpoints. The usefulness of BPV-1-induced transformation as a bioassay for detecting chemicals with promoting activities is discussed.

Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A.

Fishermen from Kerala (India) who chewed tobacco-containing betel quids daily (17.2 +/- 9.6 quids per day) and had well-developed oral leukoplakias with elevated frequencies of micronucleated cells participated in a short-term intervention trial. Beta-carotene (180 mg/week) (Group I), beta-carotene (180 mg/week) plus vitamin A (100,000 IU/week) (Group II), and placebo (Group III) capsules were given twice weekly for 6 months under strict supervision. The remission of oral leukoplakias, the inhibition of new leukoplakias, and the reduction of micronucleated oral mucosal cells were recorded at the 3rd and 6th months of the trial period. After 3 months, the frequency of micronucleated cells was significantly reduced in Group I (from 4.09% to 1.1% in areas of leukoplakia, and from 4.1% to 1.0% in the normal mucosa). At this time, remission of oral leukoplakias did not differ significantly from that observed in the placebo group. After 6 months of treatment, remission of leukoplakias in Group I (14.8%) and Group II (27.5%) differed significantly from that seen in Group III (3.0%). The development of new leukoplakias during the 6-month period was strongly inhibited in Group II (7.8%), and to a lesser degree in Group I (14.8%), as compared to Group III (21.2%). During the trial period, all participants continued to chew tobacco-containing betel quids in their accustomed manner. Thus, remission and inhibition of new oral leukoplakias and reduction of micronucleated mucosal cells occurred in the groups receiving beta-carotene and beta-carotene plus vitamin A during the continuous presence of carcinogens derived from tobacco and areca nut.

Effect of retinoic acid on bovine papillomavirus (BPV) DNA-induced transformation and number of BPV DNA copies.

The effect of all-trans-retinoic acid (RA) was examined on (1) transformation induced in C127 cells by transfection with plasmid pdBPV-1 (142-6), which contains DNA of bovine papillomavirus (BPV), (2) the capacity of transformed BPV DNA-containing clones to form colonies with transformed properties (e.g., piling up into multilayered colonies), and (3) the number of BPV DNA copies in transformed cells. At nontoxic doses ranging from 10(-7) to 10(-5) M, RA reduced the frequency of transformed foci in a dose-dependent manner. The extent of inhibition depended on the length of RA treatment and on the time that elapsed between DNA transfection and RA treatment. RA exerted only a slight inhibitory effect during the first days after transfection. Complete inhibition was observed when the cells were continuously exposed after transfection to RA at doses ranging from 0.5 to 1 X 10(-5) M. The inhibitory effect of RA on transformation was reversible. Transformed foci started to form after withdrawal of RA treatment. In the presence of RA (5 X 10(-6) M), cells from transformed colonies were no longer able to form foci displaying transformed properties. The number of BPV DNA copies gradually decreased when the cells were grown over several generations in the presence of RA (5 X 10(-6) M). After approximately 30 cell generations, the cell cultures appeared to have less than one copy of BPV DNA.