Carcinogen-induced Damage Research Articles

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Cancer-preventing attributes of probiotics: an update

Cancer is a serious global public health problem. Cancer incidence and mortality have been steadily rising throughout the past century in most places of the world. There are several epidemiological evidences that support a protective role of probiotics against cancer. Lactic acid bacteria and their probioactive cellular substances exert many beneficial effects in the gastrointestinal tract, and also release various enzymes into the intestinal lumen and exert potential synergistic (LAB) effects on digestion and alleviate symptoms of intestinal malabsorption. Consumption of fermented dairy products with LAB may elicit anti-tumor effects. These effects are attributed to the inhibition of mutagenic activity, the decrease in several enzymes implicated in the generation of carcinogens, mutagens, or tumor-promoting agents, suppression of tumors, and epidemiology correlating dietary regimes and cancer. Specific cellular components in lactic acid bacteria seem to induce strong adjuvant effects including modulation of cell-mediated immune responses, activation of the reticulo-endothelial system, augmentation of cytokine pathways, and regulation of interleukins and tumor necrosis factors. Studies on the effect of probiotic consumption on cancer appear promising, since recent in vitro and in vivo studies have indicated that probiotic bacteria might reduce the risk, incidence and number of tumors of the colon, liver and bladder. The protective effect against cancer development may be ascribed to binding of mutagens by intestinal bacteria, may suppress the growth of bacteria that convert procarcinogens into carcinogens, thereby reducing the amount of carcinogens in the intestine, reduction of the enzymes β-glucuronidase and β-glucosidase and deconjugation of bile acids, or merely by enhancing the immune system of the host. There are isolated reports citing that administration of LAB results in increased activity of anti-oxidative enzymes or by modulating circulatory oxidative stress that protects cells against carcinogen-induced damage. These include glutathione-S-transferase, glutathione, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase. However, there is no direct experimental evidence for cancer suppression in human subjects as a result of the consumption of probiotic cultures in fermented or unfermented dairy products, but there is a wealth of indirect evidence based largely on laboratory studies.

Hepatoprotective activity of bacoside A against N-nitrosodiethylamine-induced liver toxicity in adult rats

N-Nitrosodiethylamine (DEN) is a notorious carcinogen, present in many environmental factors. DEN induces oxidative stress and cellular injury due to enhanced generation of reactive oxygen species; free radical scavengers protect the membranes from DEN-induced damage. The present study was designed to evaluate the protective effect of bacoside A (the active principle isolated from Bacopa monniera Linn.) on carcinogen-induced damage in rat liver. Adult male albino rats were pretreated with 15 mg/kg body weight/day of bacoside A orally (for 14 days) and then intoxicated with single necrogenic dose of N-nitrosodiethylamine (200 mg/kg bodyweight, intraperitonially) and maintained for 7 days. The liver weight, lipid peroxidation (LPO), and activity of serum marker enzymes (aspartate transaminases, alanine transaminases, lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transpeptidase) were markedly increased in carcinogen-administered rats, whereas the activities of marker enzymes were near normal in bacoside A-pretreated rats. Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutatione-S-transferase, and reduced glutathione) in liver also decreased in carcinogen-administered rats, which were significantly elevated in bacoside A-pretreated rats. It is concluded that pretreatment of bacoside A prevents the elevation of LPO and activity of serum marker enzymes and maintains the antioxidant system and thus protects the rats from DEN-induced hepatotoxicity.

Assessment of glutathione-S-transferase and glutathione reductase in patients with squamous-cell carcinoma of buccal mucosa.

Serum and tumor cytosolic levels of glutathione-S-transferase (GST) and glutathione-reductase (GR) activity were determined spectrophotometrically. The levels were correlated with clinicopathological criteria and a tobacco-associated protein band (T band) found in serum. The results showed significantly decreased mean serum GST levels (p < 0.02) in cancer patients as compared with controls. However, mean serum GR levels were significantly higher in patients than in controls (p < 0.01). T-band-positive patients showed low GST and low GR activity as compared with T-band-negative patients. Tumor cytosolic-enzyme levels of GST and GR activity were significantly higher (p < 0.0003 and p < 0.0001, respectively) than in corresponding adjacent noncancerous mucosa. Tumour cytosolic GST and GR activity showed significant association with clinicopathologic criteria, e.g., stage, histologic grade and nodal involvement. T-band-negative patients showed significantly higher levels of GST (p < 0.0001) than did T-band-positive patients. Low levels of cytosolic GST may be associated with increased susceptibility towards carcinogen-induced damage. The results suggest that the presence of T band in the sera may be associated with a high-risk phenotype due to decreased detoxification ability.

Sequence of the FRA3B common fragile region: implications for the mechanism of FHIT deletion.

The hypothesis that chromosomal fragile sites may be "weak links" that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the FHIT gene, which encompasses the common fragile site, FRA3B. Sequence analysis of 276 kb of the FRA3B/FHIT locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in FHIT gene internal deletions, probably as a result of carcinogen-induced damage at FRA3B fragile sites.

A New Look at the Bioactive Properties of Anthocyanin-rich Horticultural Crops

The bright red pigmentation in edible, anthocyanin-rich fruits and vegetables is a definite bonus in terms of market appeal. As a result, breeders have worked consistently to intensify anthocyanin levels or alter composition in crops. The positive links between consumption of crops and food products containing natural anthocyanin pigments, and reduced incidence of cardiovascular and other chronic diseases, have been established anecdotally and more recently validated in research trials including those from our laboratory group. The protective events, most attributed to the potent antioxidant properties of anthocyanin pigments and associated phytochemicals, place anthocyanin-rich crops in the category of “Functional Foods,” yielding health protection unrelated to nutritional value. In vitro bioactivity assays have identified components from these crops capable of blocking the initiation stages of carcinogenesis, while a completely separate class of phytochemicals and sets of assays establish efficacy against the promotion stages of tumorigenesis. Animal models for carcinogen-induced damage to mammary gland and skin DNA subsequently demonstrate the in vivo potency of the same target compounds. Similarly, to establish cardioprotective properties, demonstrations of ability to inhibit platelet aggregation, relax vascular muscle tissue, and reduce total serum cholesterol are demonstrated in a series of in vitro assays, and via animal models and human studies. While activity-directed fractionations seek to identify specific responsible compounds, it is increasingly evident that bioactivity is drastically attenuated once specific compounds are isolated, and the synergistic interaction of associated phytochemicals in horticultural crops is prerequisite to realizing health benefits. These complications have slowed the establishment of effective minimum “dosages,” but all the more strongly promote consumption of the crops.

Genetic susceptibility to head and neck cancer: interaction between nutrition and mutagen sensitivity.

The development of head and neck cancer may depend not only on exposure to environmental carcinogens but also on a genetically based susceptibility to carcinogen-induced damage. This thesis presents a case-control study that demonstrates the significance of mutagen sensitivity, a measure of an individual's intrinsic DNA repair capacity against free radical damage, as a risk factor for the disease. As part of the case-control analysis, 167 previously untreated patients and 177 age- and sex-matched healthy controls were assessed for various lifestyle factors including tobacco and alcohol habits, occupational exposures, and diet. Mutagen sensitivity expressed by each individual was determined by quantifying bleomycin-induced chromosomal breaks within peripheral blood lymphocytes in vitro. Consistent with our initial observations and those of others, mutagen hypersensitivity was strongly associated with increased risk of head and neck cancer (odds ratio, 4.95; 95% confidence interval, 2.67 to 9.17) after adjusting for age, sex, and race. Low intake of vitamins C and E was also associated with an increased risk of disease and was interactive with mutagen sensitivity in risk estimates. Individuals with both a low intake of various antioxidants and increased chromosomal sensitivity to oxidant-induced DNA damage were at greatest risk. This study supports the concept that the risk of head and neck cancer is determined by a balance of factors that either enhance or protect against free radical oxygen damage, including innate capacities for DNA repair.

Correlation of DNA adducts in blood mononuclear cells with tobacco carcinogen-induced damage in human lung

The formation of carcinogen-DNA adducts within the respiratory epithelium is thought to be a critical factor in the induction of lung cancer from tobacco smoke. A reliable surrogate measure of carcinogen damage to the lung would be of great value in molecular epidemiological studies of cancer risk. The validity of measurements of DNA adducts formed from hydrophobic aromatic hydrocarbons in peripheral blood mononuclear cells (MNCs) was investigated by comparing the levels of aromatic DNA adducts detected in lung tissue from 31 lung cancer patients with those detected in MNCs from the same individuals using the 32P-postlabeling assay. The associations of smoking history and intake of dietary antioxidants with adduct levels also were assessed. Tissue-specific, as well as common DNA adducts were detected in lung and blood; total MNC adduct levels were highly correlated with total lung adducts. After smoking cessation, adduct levels appeared to decay in both tissues at similar rates. Multivariate analyses (Poisson regression modeling) indicated that dietary antioxidant intake (carotenoids, vitamin A, and retinol) modified the levels of aromatic DNA adducts in both the lungs and blood. Of all models tested, the optimal one for predicting lung adduct levels included the measure of blood MNC adduct levels only. Therefore, blood MNCs are a valid surrogate tissue for estimating the burden of DNA adducts in respiratory tissue in molecular epidemiological studies.

Biomonitoring human exposure to environmental carcinogenic chemicals.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.

Interaction of 7H-dibenzo[c,g]carbazole and its organspecific derivatives with hepatic mitochondrial and nuclear DNA in the mouse.

The recent observation of a high level of adducts in mitochondrial DNA (mtDNA) of cells exposed to chemical carcinogens aroused new interest in the hypothesis that carcinogen-induced damage in mitochondria plays a role in one or more stages of carcinogenesis. In order to investigate whether differences in the metabolic activation of carcinogens have qualitative and quantitative effects on ml- and nuclear DNA (nuDNA) adduct formation, mice were exposed to the potent hepatocarcinogenic and sarcomagenic polycyclic hydrocarbon 7H-dibenzo[c,g]carbazole (DBC) and to three of its derivatives that show large differences in enzymatic activation: N-acetyl-DBC (N-AcDBC), which is carcinogenic for several tissues; 5,9-dimethyl-DBC (DiMeDBC), which is exclusively hepatocarcinogenic; and N-methyl-DBC (N-MeDBC), which is exclusively sarcomagenic. Adduct formation and toxic effects were measured over 48 hr. With a moderate 5 mumol/kg dose of DBC, the adduct level in liver 24 hr after treatment was always higher in nuDNA than in mtDNA; after 48 hr a substantial increase in the level of adducts in mtDNA was observed, with a parallel decrease in the level in nuDNA. With DiMeDBC, a 4.9-fold increase in mtDNA was seen at 48 hr, whereas, at the same dose, the non-hepatocarcinogenic N-MeDBC induced a very small number of adducts. In order to obtain a nearly identical level of adducts in nu- and mtDNA at 24 hr, the dose of DBC must be three times higher (15 mumol/kg); this and higher dose levels had a strong cytotoxic effect in liver cells. Qualitative differences in adduct distribution were observed on chromatograms of mtDNA and nuDNA, showing that the access to mtDNA is a complex process. Our results confirm that mouse liver mtDNA is a major target for DBC and its hepatocarcinogenic derivatives. The possible interference of genotoxic alterations in mtDNA with carcinogenic mechanisms is discussed.

Selenium. Mechanistic aspects of anticarcinogenic action.

Selenium is increasingly recognized as a versatile anticarcinogenic agent. Its protective functions cannot be solely attributed to the action of glutathione peroxidase. Instead, selenium appears to operate by several mechanisms, depending on dosage and chemical form of selenium and the nature of the carcinogenic stress. In a major protective function, selenium is proposed to prevent the malignant transformation of cells by acting as a "redox switch" in the activation-inactivation of cellular growth factors and other functional proteins through the catalysis of oxidation-reduction reactions of critical SH groups of SS bonds. The growth-modulatory effects of selenium are dependent on the levels of intracellular GSH and the oxygen supply. In general, growth inhibition is achieved by the Se-mediated stimulation of cellular respiration. Selenium appears to inhibit the replication of tumor viruses and the activation of oncogenes by similar mechanisms. However, it may also alter carcinogen metabolism and protect DNA against carcinogen-induced damage. In additional functions of relevance to its anticarcinogenic activity, selenium acts as an acceptor of biogenic methyl groups, and is involved in the detoxification of metals and of certain xenobiotics. In its interactions with transformed cells at higher concentrations, it may induce effects ranging from metabolic and phenotypical changes, and partial renormalization to selective cytotoxicity owing to reversible or irreversible inhibition of protein and DNA synthesis. Selenium also has immunopotentiating properties. It is required for optimal macrophage and NK cell function. Its protective effects are influenced by synergistic and antagonistic dietary and environmental factors. The latter include a variety of toxic heavy metals and xenobiotic compounds, but they are also influenced by essential elements, such as zinc. The exposure to antagonistic factors must be minimized for the full expression of its anticarcinogenic potential.

Chemical carcinogen-induced damage to the c- neu and c- myc protooncogenes in rat lung epithelial cells: possible mechanisms for differential repair in transcriptionally active genes

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced damage to two transcriptionally active genes, c-neu and c-myc, was studied in rat lung epithelial cells in vitro. MNNG, a direct acting alkylating agent that produces alkalilabile sites in DNA, caused damage to both protooncogenes. DNA damage was determined by monitoring the disappearance of specific fragments generated by restriction enzyme digestion in Southern blots. DNA repair in the c-neu and c-myc protooncogenes was examined in confluent cells by measuring the reappearance of these same bands. While the c-neu gene exhibited repair in 24 h, none was detected in the c-myc gene. These results imply that the promutagenic DNA lesions caused by MNNG are differentially repaired in two transcriptionally active genes.

Binding of polycyclic and nitropolycyclic aromatic hydrocarbons to specific fractions of rat lung chromatin

Carcinogen-induced damage to nuclear matrix DNA, the site of DNA replication and transcription, could have profound effects on gene regulation and mutation. 1,6-Dinitropyrene (1,6-DNP), 1-nitropyrene (1-NP), 6-nitrobenzo[a]pyrene (6-NBP), benzo[a]pyrene (BP) and benzo[a]pyrene diolepoxide (BPDE) were investigated for their abilities to bind to selected regions of DNA in rat lung cell nuclei. Following in vitro exposure to carcinogen, nuclei were fractionated into active chromatin (AC), nuclear matrix (NM) and bulk (low and high salt) chromatin fractions. At an equivalent molar concentration, the highest binding to unfractionated (total) DNA was obtained with BPDE, followed in order by BP, 1,6-DNP, 6-NBP and 1-NP. BPDE, a direct alkylating compound, was bound ∼18 times higher than the other compounds. All compounds were bound to AC (mononucleosomal DNA ∼185 bp) and to NM in greater amounts than to bulk DNA. The binding ratios (AC + NM) (LS + HS) varied from 2 to 21, depending upon the compound. The selective binding to specific DNA regions did not appear to be significantly related to the structures of the parent compounds or to their inferred metabolites. Thus, it appears that selective binding to specific regions is a general phenomenon that is related to the open state of the chromatin structure.

Repair of chemical carcinogen-induced damage in DNA of human lymphocytes and lymphoid cell lines — Studies of the kinetics of removal of O6-methylguanine and 3-methyladenine

The removal of O 6-methylguanine by human lymphoid cells corresponded, with certain assumptions, to a second-order chemical reaction in any given cell. There was a spectrum of proficiency in this respect for a considerable number of cells originating from different individuals and it was found that patients with diseases associated with autoimmunity tended to fall into the less proficient groups. E-B virus-induced lymphoid cell lines, derived from proficient, but not relatively deficient, peripheral blood lymphocytes, always (in 9/9 cases) reflected the level of proficiency of the donor lymphocytes with respect to removal of O 6-methylguanine. Thus while proficient lymphocytes always produced proficient cell lines, deficient lymphocytes, in 3/8 cases, gave rise to more proficient cell lines. No evidence was found that groups of individuals exist who lack ability to remove 3-methyladenine from DNA, either from their blood lymphocytes or derived lymphoid cell lines.

The frequency and distribution of apoptosis induced by three non-carcinogenic agents in mouse colonic crypts

The appearance of apoptotic cells has been used as an index of carcinogen-induced damage inflicted on the colonic epithelium. We report here that 3 potent biological toxins, colchicine, puromycin and diphtheria toxin which have not been shown to be carcinogenic cause a dose-related increase in apoptotic cells in the colonic epithelium of the mouse. The distribution of apoptosis along the crypt length is characteristic for each of the agents studied.

Diurnal variation in the response of gamma-ray-induced apoptosis in the mouse intestinal epithelium

The appearance of apoptosis has been used as an index of carcinogeninduced damage incurred by the intestinal epithelium. Here we report that the cytotoxic response elicited by gamma radiation in the duodenal and colonic epithelium of the mouse is markedly affected by the time of day at which the animals are irradiated or sampled. The diurnal pattern in radiosensitivity of the crypt cells, as measured by the appearance of apoptosis, is reflective of circadian rhythms in mouse intestinal cells. Circadian effects should therefore be considered when interpreting apoptosis data obtained at one time point only or when comparing data obtained at different times of the day.

Evidence of carcinogen-induced replication of partially-repaired DNA in target cells during nitrosamine carcinogenesis

Damage and repair of rat kidney DNA, following administration of the renal carcinogens dimethylnitrosamine and diethylnitrosamine, has been analysed by chromatography on benzoylated-DEAE-cellulose. For this purpose, DNA was labelled in vivo by administration of 3 H-thymidine 30 thr after injection of folic acid. After a recovery period of 10 days, single doses of nitrosamines were administered by i.p. injection. Both compounds caused a dose-dependent increase in the proportion of renal DNA bound to benzoylated-DEAE-cellulose due to the presence of single-stranded regions. In the case of dimethylnitrosamine, greatest structural damage of DNA was detected after completion of the alkylation reaction in vivo. The recovery of renal DNA from nitrosamine-induced damage was monitored in a sequential study. With dimethylnitrosamine, a biphasic pattern was recorded, maximal damage to DNA being evident 1 and 4 days after administration of the carcinogen. The increase at 4 days may be attributed to dimethylnitrosamine-induced proliferative activity in the kidney. The data are discussed with reference to the role of replication of cells containing persistent carcinogen-induced damage in this tumour induction system.

Damage of proliferating lymphoid cell deoxyribonucleic acid by methyl methanesulfonate and N-acetoxy-2-acetylaminofluorene

Replicative synthesis of deoxyribonucleic acid by splenic lymphoid cells cultured from C57BL/6J strain mice and stimulated to proliferate with concanavalin A (Con A) was strongly suppressed by short exposure (10-60 min) of the cells to the direct-acting carcinogens methyl methanesulfonate or N-acetoxy-2-acetylaminofluorene. Carcinogen-treated lymphoid cells failed to recover stimulated levels of replicative synthesis after incubation for several hours in fresh medium without carcinogen. In contrast, spleen cells treated with hydroxyurea (a metabolic inhibitor of deoxyribonucleic acid replication) fully recovered stimulated rates of replicative synthesis upon incubation in medium without hydroxyurea. Furthermore, the inhibition of replicative synthesis by the carcinogens was accompanied by marked stimulation of deoxyribonucleic acid repair synthesis. Concomitant measurement of replicative and repair synthesis appears to be an effective approach for the in vitro study of carcinogen-induced damage in the deoxyribonucleic acid of proliferating lymphoid cells.

DNA phosphotriesters as indicators of cumulative carcinogen-induced damage.

RECENT studies on phosphotriesters induced in DNA in vivo by several potent chemical carcinogens have suggested that these lesions are chemically stable and that they are not eliminated by cellular DNA-repair systems2–3. It seemed possible therefore that in long-term carcinogenesis studies the accumulation of phosphotriesters might be used as a measure of the extent of damage to DNA. In a preliminary study rats have been fed continuously with either dimethylnitrosamine or diethylnitrosamine in the drinking water (0.1–5 p.p.m., w/v) and significant concentrations of methyl or ethyl phosphotriesters have been detected in tissue DNA even at the lowest dose.

Effect of alkane tumor-promoting agents on chemically induced mutagenesis in cultured V79 Chinese hamster cells.

Linear alkanes of specific chain length between 6 and 16 carbon atoms, an aryl derivative of dodecane, and a phorbol diester were tested in a cell culture system for relative ability to enhance mutagenesis induced by a chemical carcinogen, methylazoxymethanol acetate (MAM). Mutation frequencies at the ouabain-resistance locus were measured. Results indicated an excellent correlation between the relative activities of the above compounds in enhancing mutagenesis in the in vitro culture system and their tumor-promoting activities in mouse skin. None of the compounds tested showed mutagenic activity per se, further lending support to the theory that promoters act via derepression of latent carcinogen-induced damage to the genome.