Mechanisms of T cell evasion by Epstein-Barr virus and implications for tumor survival.

Epstein-Barr virus (EBV) is a prevalent oncogenic virus estimated to infect greater than 90% of the world's population. Following initial infection, it establishes latency in host B cells. EBV has developed a multitude of techniques to avoid detection by the host immune system and establish lifelong infection. T cells, as important contributors to cell-mediated immunity, make an attractive target for these immunoevasive strategies. Indeed, EBV has evolved numerous mechanisms to modulate T cell responses. For example, it can augment expression of programmed cell death ligand-1 (PD-L1), which inhibits T cell function, and downregulates the interferon response, which has a strong impact on T cell regulation. It also modulates interleukin secretion and can influence major histocompatibility complex (MHC) expression and presentation. In addition to facilitating persistent EBV infection, these immunoregulatory mechanisms have significant implications for evasion of the immune response by tumor cells. This review dissects the mechanisms through which EBV avoids detection by host T cells and discusses how these mechanisms play into tumor survival. It concludes with an overview of cancer treatments targeting T cells in the setting of EBV-associated malignancy.

Seasonal variations in the levels of PAH-DNA adducts in young adults living in Mexico City.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous components of polluted air. The Mexico City Metropolitan Area (MCMA), one of the most densely populated areas in the world, is 2240 m above sea level. At this altitude, less oxygen is available, making combustion less efficient and therefore producing more PAH pollutants. According to the Automatic Monitoring Network in Mexico City (RAMA, for its Spanish initials; http://www.sma.df.gob.mx/simat2/informaciontecnica/index.php?opcion=5&opciondifusion_bd=90), which performs environmental monitoring, the critical air pollutants in Mexico City are ozone and particulate matter (PM). PM emissions increase during the dry season (winter to spring) and decrease during the rainy season (summer to autumn). The bioactivation of some PAHs produces reactive metabolites that bind to DNA, and the presence of elevated levels of PAH-DNA adducts in tissues such as blood lymphocytes represents an elevated risk for the development of cancer. We have compared the levels of PAH-DNA adducts and the percentage of cells with chromosomal aberrations (CWAs) using a matched set of peripheral blood lymphocytes obtained on two separate occasions from young non-smoking inhabitants of the MCMA (n = 92) during the 2006 dry season and the following rainy season. PAH-DNA adducts were analysed using the r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay (CIA). The percentages of CWA were determined in cultured lymphocytes from the same individuals. Both DNA adduct levels and chromosomal aberrations were tested for correlation with lifestyle and the polymorphisms of cytochromes P450 CYP1A1 and CYP1B1 as well as glutathione-S-transferases GSTM1 and GSTT1. The levels of PAH-DNA adducts were significantly higher (P < 0.001) in the dry season (10.66 ± 3.05 per 10(9) nt, n = 92) than during the rainy season (9.50 ± 2.85 per 10(9) nt, n = 92) and correlated with the seasonal levels of particulate matter with a diameter of ≤ 10 µm (PM(10)). The percentage of CWA was not seasonally related; however, significant associations between the number of risk alleles and adduct levels in the dry (R = 0.298, P = 0.048) and in the wet seasons (R = 0.473, P = 0.001) were observed.

Nuclear bud formation: a novel manifestation of Zidovudine genotoxicity.

Normal diploid somatic mammalian cell division generates 2 daughter cells as a result of a strict and well-controlled mitotic process. However, some defects during the progression of that process could generate an unbalanced distribution of chromosomes, aneuploidy and eventually, a malignant phenotype. Previous observations using a transgenic mouse model with diminished DNA repair capacity revealed the presence of nuclear buds (NBs) induced in vitro by the nucleoside analog zidovudine (Retrovir(R), 3'-azido-3'-deoxythymidine, AZT). Here we used bone marrow mesenchymal cells, taken from mice with the Xpa(-/-)Trp53(+/-) genotype, that were cultured and exposed to 0 and 100 muM AZT for 24 hours. Fixed and denatured cells were processed by fluorescence in situ hybridization (FISH) with whole chromosome painting probes used to identify chromosomes in cells growing on glass chamber slides (2 probes/slide). A variety of sizes and shapes of NBs were observed. Some NBs had a large connection with the main nucleus (>(1/4) of the NB diameter), others hada smaller connection (<(1/4) of the NB diameter), some were circular and positioned close to the nucleus, while some resided in the cytoplasm separated from the nucleus or connected by a thin chromatin strand. We had hypothesized that NBs would progress in the process of budding until separation occurred, but this was not proven by time-lapse photography studies performed for 20 hours. From 1,126 cells scored in the unexposed cultures, 10.39 % of cells carried NBs, while from 1,108 cells scored in the AZT-exposed cultures 29.16% of cells carried NBs (p = 0.001). In AZT-exposed cells there were a total of 322 NBs scored; 46.6% or 150 NBs contained positive signals for one or both probes used, while 53% or 172 NBs had no probe signal. In addition, FISH analysis showed no preferential localization of any chromosome within the NBs. Among the NBs that carried no probe signal, the presence of positive signals with inversion of DAPI imaging demonstrated centromeric content. It has been hypothesized that NBs occur as a result of expulsion of amplified DNA from the main nucleus; however, this data demonstrates that NBs may contain any chromosome, suggesting that NBs do not consist of just amplified DNA.

Quantitation of cis-diamminedichloroplatinum II (cisplatin)-DNA-intrastrand adducts in testicular and ovarian cancer patients receiving cisplatin chemotherapy.

The antitumor activity of cis-diamminedichloroplatinum II (cisplatin) is believed to be related to its covalent interaction with DNA where a major DNA binding product is an intrastrand N7-bidentate adduct on adjacent deoxyguanosines. A novel immunoassay was used to quantitate this adduct in buffy coat DNA from testicular and ovarian cancer patients undergoing cisplatin therapy. 44 out of 120 samples taken from 45 cisplatin patients had detectable cisplatin-DNA adducts. No adducts were detected in 18 samples of DNA taken from normal controls, patients on other chemotherapy, or patients before treatment. The quantity of measurable adducts increased as a function of cumulative dose of cisplatin. This was observed both during repeated daily infusion of the drug and over long-term, repeated 21-28 d cycles of administration. These results suggested that adduct removal is slow even though the tissue has a relatively rapid turnover. Patients receiving cisplatin for the first time on 56-d cycles, and those given high doses of cisplatin as a "salvage" regimen, did not accumulate adducts as rapidly as patients on first time chemotherapy on 21- or 28-d cycles. Disease response data, evaluated for 33 cisplatin-treated patients, showed a positive correlation between the formation of DNA adducts and response to drug therapy. However, more data will be required to confirm this relationship. These data show that specific immunological probes can readily be applied to quantitate DNA adducts in patients undergoing cancer chemotherapy.

Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA.

BACKGROUND: Epidermal growth factor receptor (EGF-R) perturbation by receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells. This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R. PURPOSE: Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity. METHODS: MDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact[p]-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA. EGF-R content was assessed by 125I-EGF binding and EGF-R immunoblot assays. Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ. Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA. RESULTS: Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both 125I-EGF binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells. By use of a colony-forming assay, the 1-hour IC50 (i.e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 microM or less for parental and vector-transfected clones (n = 4), whereas it was 25 microM or more for all MDA-468/AS-EGFR clones (n = 3). MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G2 arrest 48 hours after a 1-hour incubation with 3-30 microM cisplatin. Under these conditions, apoptosis and G2 arrest were undetectable in all MDA-468/AS-EGFR clones. An MDA-468 subline selected after long-term treatment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: > 30 microM) compared with parental cells. This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells. Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones. CONCLUSIONS: These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA.

Enhanced malignant conversion of benign mouse skin tumors by cisplatin.

The chemotherapeutic agent cisplatin, reported to be a complete carcinogen in rodents and a tumor initiator for mouse skin, was tested for activity to enhance the conversion of carcinogen-induced skin papillomas to carcinomas. Initiation of mouse skin by 7,12-dimethylbenz[a]anthracene followed by 12 weeks of promotion by 12-O-tetradecanoylphorbol-13-acetate produced seven to eight papillomas/mouse. Ten weekly injections of 100 micrograms of cisplatin into these papilloma-bearing mice induced a 2.3-fold enhancement of conversion relative to the spontaneous rate of 1.9%. Even a single exposure to cisplatin in tumor-bearing mice increased the carcinoma incidence to the same extent as 10 exposures to urethane, an agent previously shown to enhance malignant conversion. At the dose tested, cisplatin was inactive as a complete carcinogen or a tumor promoter. Cisplatin-DNA adducts, measured in samples from skin, liver, and kidneys, were persistent for at least 4 weeks after the last exposure to cisplatin. Thus cisplatin is a relatively potent inducer of the putative genotoxic changes required for conversion of skin tumors from a benign to a malignant phenotype. The activity of cisplatin in the initiation and malignant conversion stages in this animal model for carcinogenesis suggests that patients given cisplatin-based chemotherapy are at increased risk for the development of treatment-induced second cancers.

DNA adducts, protein adducts, and sister chromatid exchange in cigarette smokers and nonsmokers.

In order to validate markers of internal dose and biologically effective dose of carcinogens, a battery of measurements was made on blood samples from 22 smokers and 24 nonsmokers. The markers included immunoreactivity in an enzyme-linked immunosorbent assay (ELISA) quantified in white blood cells with the use of a polyclonal anti-benzo[a]pyrene diol epoxide-I-DNA antibody, 4-aminobiphenyl hemoglobin (4-ABP-Hb) adducts measured by negative chemical ionization mass spectrometry, sister chromatid exchange (SCE) in cultured lymphocytes, and cotinine in plasma measured by radioimmunoassay. Several blood samples were drawn from each subject. In blood samples 1 and 3 having detectable levels of DNA adducts, mean femtomole-per-microgram levels were consistently higher among smokers compared to nonsmokers. The borderline significance of this difference may be attributable to the small numbers of subjects. Consistently higher adduct levels were seen in females compared to males. In sample 3, adduct levels were significantly correlated with measurements of active smoking in smokers and with passive smoking in nonsmokers. By contrast to the ELISA data, which may reflect cumulative exposure from multiple background sources, the 4-ABP-Hb assay was able to distinguish clearly between smokers and nonsmokers. SCEs were significantly elevated in the smokers compared to nonsmokers. Also observed were significant correlations between 4-ABP-Hb and both cotinine and SCEs, as well as a positive correlation between the 4-ABP-Hb and DNA adduct levels (sample 3) that was highly significant. The correlation between DNA and 4-ABP-Hb adducts was significant in smokers but not nonsmokers (sample 3). These results support the need for batteries of markers to detect and to quantify the carcinogenic dose to humans resulting from both specific and "background" environmental exposures.

Transplacental effects of 3'-azido-2',3'-dideoxythymidine (AZT): tumorigenicity in mice and genotoxicity in mice and monkeys.

BACKGROUND: When given during pregnancy, the drug 3'-azido-2',3'-dideoxythymidine (AZT) substantially reduces maternal-fetal transmission of human immunodeficiency virus type 1 (HIV-1). However, AZT has been shown to be carcinogenic in adult mice after lifetime oral administration. In this study, we assessed the transplacental tumorigenic and genotoxic effects of AZT in the offspring of CD-1 mice and Erythrocebus patas monkeys given AZT orally during pregnancy. METHODS: Pregnant mice were given daily doses of either 12.5 or 25.0 mg AZT on days 12 through 18 of gestation (last 37% of gestation period). Pregnant monkeys were given a daily dose of 10.0 mg AZT 5 days a week for the last 9.5-10 weeks of gestation (final 41%-43% of gestation period). AZT incorporation into nuclear and mitochondrial DNA and the length of chromosomal end (telomere) DNA were examined in multiple tissues of newborn mice and fetal monkeys. Additional mice were followed from birth and received no further treatment until subjected to necropsy and complete pathologic examination at 1 year of age. An anti-AZT radioimmunoassay was used to monitor AZT incorporation into DNA. RESULTS: At 1 year of age, the offspring of AZT-treated mice exhibited statistically significant, dose-dependent increases in tumor incidence and tumor multiplicity in the lungs, liver, and female reproductive organs. AZT incorporation into nuclear and mitochondrial DNA was detected in multiple organs of transplacentally exposed mice and monkeys. Shorter chromosomal telomeres were detected in liver and brain tissues from most AZT-exposed newborn mice but not in tissues from fetal monkeys. CONCLUSIONS: AZT is genotoxic in fetal mice and monkeys and is a moderately strong transplacental carcinogen in mice examined at 1 year of age. Careful long-term follow-up of AZT-exposed children would seem to be appropriate.

Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay.

Rabbit antiserum prepared against N-(guanosin-8-yl)-acetylaminofluorene was utilized in radioimmunoassay to detect formation and removal of C-8 adducts from the DNA of cultured cells exposed to N-acetoxy-2-acetylaminoflorene. The assay was able to quantitate both acetylated and deacetylated C-8 adducts between 0.5 and 5 pmol while the N2 adduct, 3-(deoxyguanosin-N2-yl)acetylaminofluorene, was not detected below 160 pmol. By varying the proportions of acetylated and deacetylated C-8 adducts in the radioimmunoassay, a series of standard curves were developed from which the relative proportion of each adduct could be determined in unknown mixtures. DNA from mouse epidermal cells and human skin fibroblasts exposed to N-acetoxy-2-acetylaminofluorene in culture contained only 3 and 5% respectively, of the C-8 adduct in the acetylated form. Quantitation by radioimmunoassay of total C-8 adducts bound to DNA yielded values approximately 25% lower than total carcinogen binding determined by radiolabeling. When removal of C-8 adducts was followed over a 23-hr, carcinogen-free culture period, mouse and human cells removed 40 and 50%, respectively, of bound acetylated and deacetylated C-8 adducts. These studies demonstrate the versatility of radioimmunoassay as a molecular probe for studies of chemical carcinogens.

Benzo(a)pyrene-DNA adduct formation and removal in mouse epidermis in vivo and in vitro: relationship of DNA binding to initiation of skin carcinogenesis.

An antiserum specific for the major benzo(a)pyrene (BP) adduct formed with deoxyguanosine in vivo has been used by enzyme-linked immunosorbent assay to monitor the formation and removal of DNA-bound products in BALB/c mouse epidermis exposed topically to initiating doses of BP and in BALB/c mouse keratinocytes exposed in vitro to BP or its activated derivatives. In mouse epidermal DNA, formation of antibody-recognizable products increased proportionally between doses of 50 and 250 nmol of BP, giving 2.3 to 6.0 fmol/micrograms of DNA, respectively, and reached a plateau of 10 to 11 fmol/micrograms of DNA at doses between 1000 and 1500 nmol. Antibody-recognizable adducts comprised roughly one-half of the total BP-DNA binding, since a 250-nmol dose of [3H]BP yielded 6 fmol/micrograms of DNA by enzyme-linked immunosorbent assay and 12.9 fmol/micrograms of DNA by radiolabeling. Removal of trans-(7R)-N2-(10-[7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]-yl)-deoxyguanosine adducts was monitored in epidermal DNA of mice exposed to 500 nmol of BP and, although no correction was approximated for DNA turnover in the skin, about one-half of the adducts formed by 24 hr were removed 3 days later, and only 10% remained at the end of a week. BP-DNA binding and removal were also studied in cultured mouse keratinocytes, where proliferating basal cells and terminally differentiating cells can be selectively studied by modulating the Ca2+ concentration of the medium. BP dose-response studies showed that, in cells of different maturation states, BP-DNA adduct levels were similar. Adduct formation greater than 10 to 11 fmol/micrograms (the highest obtained in vivo) was associated with extensive cytotoxicity and cell death. The kinetics of adduct removal was followed in culture under conditions in which dilution by DNA synthesis or cell loss could be monitored. Results of these experiments suggested that initial removal of BP-DNA adducts was more rapid in the differentiating population although, in both populations, 50% of the adduct was removed by 24 hr. The formation of foci resistant to Ca2+-induced terminal differentiation has been associated previously with carcinogen treatment in cultured keratinocytes. Exposure to BP or the antidiol-epoxide, at concentrations producing low cytotoxicity, yielded frequencies of differentiation-altered foci proportional to the dose of the compound used and to the number of DNA adducts formed.

Formation and removal of (guan-8-yl)-DNA-2-acetylaminofluorene adducts in liver and kidney of male rats given dietary 2-acetylaminofluorene.

Radioimmunoassay has been used to measure acetylated and deacetylated deoxyguanosine C-8 DNA adducts of 2-acetylaminofluorene in liver and kidney DNA of male Wistar-Furth rats fed a dietary regimen of 0.02 or 0.04% 2-acetylaminofluorene. When adduct formation was monitored during continuous feeding up to 60 days, substantial levels of binding (80 fmol/microgram DNA) were observed in liver by 1 day, and maximum steady-state values averaging 230 fmol/microgram DNA were reached by 30 days. Initially, during the continuous feeding, about 80% of the total C-8 adducts were deacetylated [N-deoxyguanosin(8-yl)aminofluorene], and this proportion increased to about 97% by 15 and 30 days of administration of 0.04 and 0.02% 2-acetylaminofluorene diets, respectively. Levels of C-8 adducts bound to kidney DNA in the same animals averaged 10 to 15% of the liver values with greater than or equal to 80% of these adducts in the deacetylated form. In separate experiments, rats were exposed to 2-acetylaminofluorene for 3, 7, 28, and 112 days; the carcinogen-containing diet was discontinued; and C-8 adducts were monitored during 1, 7, and 28 days of feeding control diet. At both carcinogen doses, after dietary administration for 3 or 7 days, there was a rapid decrease in liver C-8 adducts so that, after 28 days on control diet, 65 to 90% of the original adducts were no longer present in the DNA. In contrast, in animals fed 0.02% 2-acetylaminofluorene for 28 days, there were high levels (70 to 100% of the original C-8 adduct) remaining on the DNA at the end of 28 days on control diet. This apparent loss in capacity for removal of C-8 adducts is discussed in relation to biological and biochemical changes induced in the rat liver during 2-acetylaminofluorene hepatocarcinogens.

Microfluorometric determination of DNA adducts in immunofluorescent-stained liver tissue from rats fed 2-acetylaminofluorene.

The intensities of immunofluorescence in nuclei stained by an antiserum specific for the DNA adduct N-deoxyguanosin(8-yl)aminofluorene (dG-8-AF), were quantified by microfluorometry in frozen liver sections from male Fischer rats fed 2-acetylaminofluorene (AAF). Results of previous studies demonstrated that dG-8-AF is the predominant adduct (80-100%) formed in livers of rats fed AAF continuously, and that nuclei of hepatocytes and bile duct epithelial cells in rats fed AAF exhibit an adduct-specific immunofluorescence. In the present investigation, nuclear staining for dG-8-AF was quantified by microfluorometry in liver sections from male Fischer rats fed 0.02% AAF continuously for 2, 4, 8, 12, 16, 20, and 28 days. Microfluorometric determinations of the intensities of nuclear immunofluorescence staining within periportal, midzonal, and centrilobular hepatocytes and bile duct epithelial cells revealed that levels of the dG-8-AF adduct increased in these cells during AAF feeding, reaching a plateau by 12 days. However, significant differences were detected in dG-8-AF levels within cells of each lobular area. Nuclei of periportal hepatocytes exhibited the most intense immunofluorescence, nuclei of centrilobular hepatocytes and bile duct epithelial cells emitted the least intense fluorescence, and nuclei of midzonal hepatocytes exhibited an intermediate fluorescence intensity. Quantitation of whole-liver levels of the dG-8-AF adduct by RIA, after extraction of DNA, also revealed that adduct accumulation reached a plateau by 12 days of AAF feeding. Thus, similar profiles of adduct accumulation were obtained by microfluorometric analysis of immunofluorescence staining within frozen liver sections, and by RIA analysis of DNA extracted from whole livers. The periportal concentration of DNA adducts in livers of rats continuously fed a carcinogenic dose of AAF may be an important early event in AAF-induced liver tumorigenesis.

Vaginal epithelial DNA damage and expression of preneoplastic markers in mice during chronic dosing with tumorigenic levels of 3'-azido-2',3'-dideoxythymidine.

3'-Azido-2',3'-dideoxythymidine (AZT, Retrovir, zidovudine), a nucleoside analogue currently used in the therapy of acquired immunodeficiency syndrome, induces papillomas and carcinomas in vaginal epithelium of mice as a result of lifetime drug administration. In this study, female CD-1 mice were administered AZT at doses of 180, 360, and 720 micrograms/ml of drinking water for 28 days to determine whether AZT became incorporated into vaginal DNA and whether this was associated with preneoplastic changes within the target tissue. In addition, bone marrow, a target for AZT-induced cytotoxicity in mice and humans, was examined for chromosomal aberrations. A positive correlation was observed between dose level of AZT, proliferation of cells in the basal layer of vaginal epithelium, and incorporation of AZT into vaginal DNA. Incorporation of AZT into vaginal DNA was originally detected by radioimmunoassay and confirmed by immunohistochemistry. An aberrant pattern for alpha 6 integrin distribution, similar to the pattern described in skin papillomas with high risk for malignant conversion, also increased with dose in mice given AZT. Chromosomal aberrations in bone marrow increased more than 4-fold in AZT-exposed animals. The genotoxicity demonstrated by incorporation of AZT into vaginal DNA and proliferation of vaginal epithelium may play an essential part in the ability of AZT to induce abnormal differentiation in vaginal epithelium and vaginal tumorigenesis in mice.

Nonspecific inhibition of DNA repair synthesis by tumor promoters in human diploid fibroblasts damaged with N-acetoxy-2-acetylaminofluorene.

The effects of selected tumor-promoting agents and their nonpromoting analogs on DNA repair synthesis were examined in human diploid fibroblasts (WI-38) damaged with N-acetoxy-2-acetylaminofluorene. Over a range of doses, three promoters (croton oil, 12-O-tetradecanoylphorbol-13-acetate, and anthralin) were found to inhibit DNA repair synthesis while their nonpromoting analogs (phorbol and 1,8-dihydroxyanthraquinone) had little effect. Another tumor promoter, phenol, inhibited DNA repair synthesis only at very high concentrations while an analog, 4-nitrophenol, produced inhibition of DNA repair synthesis at molar concentrations at which phenol had no effect. To investigate the specificity of this phenomenon, the effects of these agents on DNA-replicative synthesis, RNA synthesis, protein synthesis, and cell morphology were evaluated. At equimolar concentrations, tumor promoters were found to inhibit DNA-replicative synthesis as effectively as repair synthesis. RNA and protein synthesis were similarly inhibited over the same range of concentrations. Extensive morphological changes, interpreted as evidence of toxicity, were seen at concentrations of promoters that inhibited the macromolecular syntheses studied. The nonpromoting analogs, with the exception of nitrophenol, had little effect on these processes and showed only slight morphological damage. Thus tumor-promoting agents appeared to inhibit a number of macromolecular synthetic events, including DNA repair synthesis. It is suggested that the effect of tumor promoters on DNA repair synthesis is part of a general response to cellular injury rather than a selective response involving a single metabolic pathway. Furthermore, it is unlikely that the inhibition of repair synthesis represents the major mode of action of promoting agents in the carcinogenic process.

Biological monitoring of fire fighters: sister chromatid exchange and polycyclic aromatic hydrocarbon-DNA adducts in peripheral blood cells.

Fire fighters are exposed to potentially carcinogenic combustion and pyrolysis products during the course of their work. The present study was designed to test 43 fire fighters and matched controls for DNA damage which might be related to occupational carcinogen exposures. Using peripheral blood lymphocytes, we examined (a) baseline sister chromatid exchange (SCE) frequency and (b) SCE induction by in vitro mutagenic challenge with mitomycin C. Using nucleated peripheral blood cells, we examined (c) polycyclic aromatic hydrocarbon-DNA adduct levels by assessing benzo(a)pyrene diol epoxide (BPDE)-DNA antigenicity. Exposures were determined from histories of fire-fighting activity. The presence of confounding factors (e.g., tobacco smoking, charcoal-broiled food consumption, etc.) was determined by questionnaire. Plasma cotinine levels were measured to assess recent exposures to tobacco smoke. White fire fighters exhibited a significantly higher risk for the presence of detectable BPDE-DNA antigenicity than white controls (odds ratio, 3.4; 95% confidence interval, 1.08-10.5 after adjustment). Consumption of charcoal-broiled food less than 3 times a month was associated with a smaller proportion of individuals exhibiting measurable (positive) BPDE-DNA antigenicity, while consumption of broiled food greater than 3 times a month did not affect the proportion of positive individuals. Daily alcohol consumption was associated with a larger proportion of individuals exhibiting positive BPDE-DNA antigenicity, (P = 0.07). Tobacco smoking and charcoal-broiled food consumption, but not fire fighting, were associated with increased levels of baseline SCE. Sensitivity to SCE induced by mitomycin C in cultured peripheral lymphocytes was similar in fire fighter and control groups. However, sensitivity of individual fire fighters to mitomycin C-induced SCE was correlated with number of fires fought in the previous 24 h.

Quantitation of benzo(a)pyrene-deoxyguanosine adducts by radioimmunoassay.

Calf thymus DNA was modified with the benzo(a)pyrene (BP) derivative, (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-) BPDE I], under conditions which yielded greater than 99% of the binding product in the form of trans-(7R)-N2-(10[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl) deoxyguanosine. Rabbits were immunized with modified DNA coupled to methylated bovine serum albumin, and the resulting antiserum was utilized in a competition radioimmunoassay for the quantitation of products of BP covalently bound to DNA. The antiserum was specific for both native and denatured immunogen DNA's as well as for the major isolated BP binding product, but it did not recognize BP, the tetrol of (+/-)BPDE I, or unmodified deoxyguanosine. The modified DNA was assayed in quantities as low as 2 pmol of adduct, a sensitivity sufficient to quantitate the extent of modification of cellular DNA when epidermal cell cultures were exposed either to BP or to (+/-)BPDE I. High-pressure liquid chromatographic analysis of DNA hydrolsates, obtained from epidermal cells exposed to BP or to (+/-)BPDE I, indicated that the major adduct was the same as than on the immunogen DNA. This approach should prove valuable for further studies on the mechanism of carcinogenesis and for monitoring human exposure to this ubiquitous carcinogen.

cis-Diamminedichloroplatinum (II)-DNA adduct formation in renal, gonadal, and tumor tissues of male and female rats.

cis-Diamminedichloroplatinum(II) (cisplatin), a potent anticancer agent, is thought to exert its cytotoxic effects through DNA damage. Using a polyclonal rabbit antisera which recognizes intrastrand bidentate deoxy(ApG)- and deoxy(GpG)-N7-diammineplatinum adducts, an enzyme-linked immunosorbent assay has been developed to quantitate this adduct in cisplatin-exposed DNA. Cisplatin-DNA adducts were measured in renal, gonadal, and tumor (sarcoma) tissues of Sprague-Dawley rats following i.v. or i.p. administration of cisplatin. When drug was administered i.v. to animals fed ad libitum adduct levels were highest in kidneys, 50% lower in s.c. sarcoma, and substantially lower in gonads. Under these experimental conditions, a large interindividual variability in adduct formation was observed in renal and tumor tissues, and adduct levels in some samples were too low to measure. Higher values among individuals were obtained using tissues of animals fasted overnight and treated i.p. Adduct levels following i.p. injections of drug were higher in kidneys and gonads of male rats than in kidneys and gonads of female rats. Analysis of tissue platinum content demonstrated higher platinum levels in kidneys of male rats than in kidneys of female rats, but the magnitude of this gender difference in total tissue platinum was not as great as that observed for adduct formation. When the influence of castration on adduct formation was investigated, adduct levels in kidneys of castrated females were higher than those in sham-operated females, but adduct levels in kidneys of the castrated male animals were not substantively different from those seen in sham-operated male controls. We conclude that the route of drug administration, diet, and hormonal status of the animal are factors that may influence cisplatin-DNA adduct formation in the rat.

Persistence of platinum-ammine-DNA adducts in gonads and kidneys of rats and multiple tissues from cancer patients.

The persistence of platinum-DNA adducts was investigated using normal rats as well as tissues from cancer patients receiving either cisdiamminedichloroplatinum(II) (cisplatin) or diamminecyclobutanedicarboxylatoplatinum(II) (carboplatin) for cancer chemotherapy. These studies used an enzyme-linked immunosorbent assay, established with a rabbit anti-cisplatin-DNA that is specific for intrastrand platinum-DNA adducts. The gonads and kidneys of male and female rats, sites for antitumor activity and toxicity, respectively, were monitored for cisplatin-DNA adduct formation after a single dose of drug and during multiple-dose exposures (once a wk for 3 wk). DNA adducts were measured by enzyme-linked immunosorbent assay 4 h and 2, 4, 7, and 14 days after administering a single i.v. injection of 8 mg/kg of cisplatin. Adduct profiles in renal tissues were similar in both males and females with adduct levels increasing between 4 h and 2 days, decreasing between Days 2 and 7, and stable between Days 7 and 14. In both sexes, levels of kidney DNA adduct measured 7 to 14 days after cisplatin injection comprised about 30% of the highest (Day 2) value. In testes and ovaries, adduct removal was complete by 4 days, and 40 to 50% of adducts present at Day 2 persisted until Days 7 and 14. A study of multiple dosing showed that adducts in renal and testicular DNA from rats given three weekly doses of 5 mg/kg of cisplatin had different accumulation profiles. In the testis there was a 2-fold accumulation of adduct after the third dose, while in the kidney adducts dropped with repeated dosing. In humans, the persistence of platinum-DNA adducts was studied in tissues from eight cancer patients who received their last dose of cisplatin or carboplatin chemotherapy between 1 day and 15 mo before autopsy. The patients had either ovarian cancer, breast cancer, or lymphoma, and the tissues studied included ovarian tumor, bone marrow, kidney, liver, spleen, lymph node, peripheral nerve, and brain. When samples were available from tumor tissues and from bone marrow within the same patient, adduct levels were similar in the two tissues. In addition, adducts were persistent for many months, since half of the individuals received their most recent platinum-drug therapy 7 to 15 mo before death. Overall, these studies demonstrate a widespread distribution and high degree of platinum-DNA adduct persistence in both animal and human tissues subsequent to cisplatin or carboplatin treatment.

Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling.

Tamoxifen (TAM), a nonsteroidal antiestrogen used as a chemotherapeutic and chemopreventive agent for breast cancer, induces liver tumors in rodents and covalent DNA adduct formation in hepatic DNA. Here, we report the development and validation of highly sensitive and specific immunoassays for the determination of TAM-DNA adducts. Rabbits were immunized with calf thymus DNA, chemically modified with alpha-acetoxytamoxifen to 2.4 adducts per 100 nucleotides, and the resulting antisera were characterized by competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemiluminescence immunoassay (CIA). Compared with DELFIA, the CIA has a much lower background and a 20-fold increase in sensitivity. For the immunogen TAM-DNA, 50% inhibition was at 2.0 +/- 0.11 (mean +/- SE, n = 18) fmol of (E)-alpha-(N2-deoxyguanosinyl)tamoxifen (TAM-dG) adduct in TAM-DNA by DELFIA. For TAM-DNA modified to 4.8 adducts in 10(6) nucleotides, 50% inhibition was at 20.6 +/- 6.6 (mean +/- SE, n = 8) fmol of TAM-dG in TAM-DNA by DELFIA and at 0.92 +/- 0.11 (mean +/- SE, n = 10) fmol of TAM-dG in TAM-DNA by CIA. No inhibition was observed in either assay with up to 20 microg (62.5 nmol of nucleotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-alpha-(N2-deoxyguanosinyl)tamoxifen and the individual compounds TAM and 4-OH-TAM gave DELFIA 50% inhibitions at 828, 2229, 5440, and 8250 fmol, respectively. For assay validation, TAM-dG levels were determined by DELFIA, CIA, and 32P-postlabeling in TAM-DNA samples modified in vitro to different levels, and comparable values were obtained in all three assays. Further validation was obtained in vivo in rat liver. DNA adducts of TAM were measurable in rat liver 24 h after a single i.p. dose of 45 mg TAM/kg body weight and after daily p.o. dosing for 7 days with 5.0, 10.0, and 20.0 mg TAM/kg body weight. In addition, TAM-DNA adducts disappeared slowly over 21 days in rats on a control diet that were first given p.o. TAM at 45 mg/kg/day for 4 days. In the rat experiments, TAM-DNA adduct levels determined by CIA compared well with those determined by 32P-postlabeling, although the CIA gave an underestimation at the highest doses. For rat liver samples, the detection limit by CIA was 3 adducts per 10(9) nucleotides (0.2 fmol of adducts per 20 microg of DNA).

Evaluation of platinum-DNA adduct levels relative to known prognostic variables in a cohort of ovarian cancer patients.

Single-agent chemotherapy with cisplatin or carboplatin can induce remissions in approximately 30% of previously treated patients with advanced stage ovarian cancer. Previous studies have shown that the extent of platinum-DNA adduct formation measured in WBC DNA of ovarian cancer patients treated with cisplatin or carboplatin is directly associated with disease response (Reed et al., Proc. Natl. Acad. Sci. USA, 84: 5024-5028, 1987). It has been unclear whether adduct level in WBC DNA is independent of known prognostic variables in this disease, or whether adduct level parallels a known prognostic variable that can be more easily monitored. In a cohort of 24 ovarian cancer patients treated with single-agent cisplatin or carboplatin, we retrospectively assessed the relationship between disease response, platinum-DNA adducts in WBC DNA, and each of eight prognostic variables by both univariate analysis and multivariate analysis. The prognostic variables evaluated included: response to previous treatment, Karnofsky status, total platinum dose prior to current therapy, stage of disease, age, bulk of disease at initiation of therapy, histological type, and histological grade. By univariate analysis, adduct level was strongly associated with disease response (two-sided P = 0.0058), with the next strongest associations with disease response being held by Karnofsky status (P = 0.125), stage of disease (P = 0.189), response to previous treatment (P = 0.352), total previous platinum dose (P = 0.358), and age (P = 0.374). No significant associations were found between adduct level and histological type or histological grade. Further, when patients were stratified by the number of cycles studied (one cycle, two cycles, or three cycles), higher levels of adduct were consistently seen in those patients responding to therapy. We conclude that, in this small cohort of refractory ovarian cancer patients treated with single-agent cisplatin or carboplatin, adduct level in WBC DNA appears to be more closely related to disease response than other previously identified prognostic variables.