Helicobacter pylori infection and antioxidants can modulate the genotoxic effects of heterocyclic amines in gastric mucosa cells

Helicobacter pylori (H. pylori) infection plays an important role in gastric carcinogenesis. This bacterium may induce cancer transformation and change the susceptibility of gastric mucosa cells to various exogenous dietary irritants. The aim of the study was to evaluate the influence of H. pylori infection on the reaction of the stomach cells to a genotoxic effect of heterocyclic amines (HCAs). These well-known mutagens are formed during cooking of protein-rich foods, primarily meat. Taking into account that persons consuming a mixed-western diet are exposed to these compound nearly an entire lifetime and more than half of human population is infected with H. pylori, it is important to assess the combined effect of H. pylori infection and HCAs in the context of DNA damage in gastric mucosa cells, which is a prerequisite to cancer transformation. We employed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) because these substances are present in a great amount in cooked and fried meat. Using alkaline comet assay, we showed that the extent of the DNA damage induced by HCAs was significantly higher in H. pylori infected gastric mucosa cells than in non-infected counterparts. We did not observed any difference in the efficiency of repair of DNA lesions induced by HCAs in both type of cells. Vitamin C reduced the genotoxic effects of HCAs in H. pylori infected and non-infected gastric mucosa cells. Melatonin more effectively decreased DNA damage caused by HCAs in H. pylori infected gastric mucosa cells as compared with control. Our results suggest that H. pylori infection may influence the susceptibility of gastric mucosa cells to HCAs and dietary antioxidative substances, including vitamin C and melatonin may inhibit the genotoxic effects of HCAs on gastric mucosa cells and may reduce the risk of carcinogenesis caused by food borne mutagens and H. pylori infection.     

LacI transgenic animal study: relationships among DNA-adduct levels, mutant frequencies and cancer incidences

In the processes of carcinogenesis caused by genotoxic carcinogens, DNA-adduct formation and resultant genetic changes are crucially important. In this report, the relationship between DNA-adduct levels and mutant frequencies (MFs), DNA-adduct levels and cancer incidences, and MFs and cancer incidences induced by heterocyclic amines (HCAs), to which humans are exposed on daily basis were investigated. There was no direct correlation between adduct levels and MFs detected after feeding Big Blue mice with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), in a comparison among various organs. Further, there was no direct correlation between DNA-adduct levels and cancer incidences, in a comparison among various organs of F344 rats. Since DNA-adducts are fixed as mutations after cell proliferation, and mutations in cancer-related genes result in cancer development, it is expected that MFs directly correlate with cancer incidences. However, there was no direct correlation between MFs and cancer incidences. Possible mechanisms involved in the discordance between DNA damage markers and cancer incidences are discussed.     

A new approach to risk estimation of food-borne carcinogens--heterocyclic amines--based on molecular information

Identification of causative agents for human cancers is the goal of our studies. We analyzed ordinary foods for mutagenicity, using the well-established Salmonella test. Heating fish and meat yielded mutagens that require metabolic activation for exhibition of mutagenicity. Structural determination revealed these mutagens to be heterocyclic amines (HCAs), their precursors in some cases being creatin(in)e, sugars and amino acids. Ten HCAs so far examined have all proved carcinogenic in mice and rats, inducing cancers in various organs such as in the mammary glands, prostate, lung, colon, skin, bladder and liver. Human exposure to HCAs is 0.1-12 microg/day, predominantly to 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). For these types of genotoxic carcinogens, DNA-adduct formation is crucially important and PhIP-DNA adducts have been detected in human tissues. However, the amounts of individual HCAs ingested by humans may not be sufficient to induce cancers by themselves and many environmental factors have also been implicated in neoplasia in man, with other considerable inter-individual variation in susceptibility, e.g., to colon carcinogenesis. This is in line with results obtained by feeding different strains of rats with HCA. Studies using lacI transgenic mice and rats have revealed that DNA adducts do not directly correlate with mutant frequencies at the organ level, or cancer incidence. However, sequencing of the Apc gene of rat colon tumors induced by PhIP revealed that it induces a signature mutation of G deletion from the GGGA sequence. This type of mutation is found in the p53 gene of 0.3% human cancers having p53-somatic mutations, and it has been calculated that 3%-10% of the p53 mutations detected in human cancers could be ascribable to PhIP. Although there remains the possibility that other carcinogens involved in human carcinogenesis cause the same signature mutation, the available data point to an important role for PhIP.     

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PK_cs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.     

Correction of DNA methylation disorder as a possible way to modulation of malignant cells drug resistance

The role of DNA methylation in functioning of a normal and cancer cell is shown in this review. The value of methylation in functioning of the systems which are involved in the process of formation of malignant cells drug resistance phenotype (regulator proteins of cellular cycle, DNA-adducts reparation, transport systems, systems of detoxication and adhesion) is explicated. The prospects of the antineoplastic therapy directed to regulation of DNA methylation by means of homocysteine and demethylation agents are analyzed.     

Carcinogenic and nephrotoxic alkaloids aristolochic acids upon activation by NADPH : cytochrome P450 reductase form adducts found in DNA of patients with Chinese herbs nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been found to be implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), and associated with the development of urothelial cancer in CHN patients. Understanding, which enzymes are involved in AA activation and/or detoxication is important in the assessment of individual susceptibility of humans to this natural carcinogen. Using the nuclease P1 version of the 32P-postlabeling assay we examined the ability of microsomal NADPH: CYP reductase to activate AA to metabolites forming DNA adducts. Renal and hepatic microsomes, containing NADPH:CYP reductase, generated AA-DNA adduct patterns reproducing those found in renal tissues in patients suffering from a renal fibrosis CHN and urothelial cancer. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two AA-DNA adducts, 7-(deoxyguanosin-N2-yl) aristolactam II and 7- (deoxyadenosin-N6-yl) aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of NADPH: CYP reductase as activating enzyme in microsomes has been proved with different cofactors and an enzyme inhibitor. Alpha-lipoic acid, a selective inhibitor of NADPH: CYP reductase, significantly decreased the amount of the adducts formed by microsomes. Likewise, only a cofactor of the enzyme, NADPH, supported the DNA adduct formation of AAI and AAII, while NADH was ineffective. These results demonstrate an involvement of NADPH: CYP reductase in the activation pathway of AAI and AAII in the microsomal system. Moreover, using the purified enzyme, the participation of this enzyme in the formation of AA-DNA adducts was confirmed. The results presented here are the first report demonstrating a reductive activation of natural nitroaromatic compounds, AA, by NADPH: CYP reductase.     

Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.     

Protective role of glutathione and glutathione transferases in mutagenesis and carcinogenesis

Glutathione (GSH) alone detoxifies electrophiles with an effectiveness which depends on the rate of the reaction and the concentration of GSH. If electrophiles are substrates for GSH transferase isoenzymes, the effectiveness of detoxication is much enhanced due to the increased rate of reaction and it is also independent of GSH concentration to low levels of GSH depletion, since the Km for GSH is approximately 0.1 mM. In this paper detoxication of electrophilic metabolites of the hepatocarcinogen N-methyl-4-aminoazobenzene which are not substrates for GSH transferases and the carcinogenic electrophile derived from the hepatocarcinogen aflatoxin B1 which is a poor substrate is compared with detoxication of electrophiles which are good substrates and which although bacterial mutagens are not carcinogenic in organs containing the appropriate GSH transferases. GSH transferases detoxify not only electrophiles derived from xenobiotics, but also endogenous electrophiles which are usually the consequence of free radical damage in the presence of oxygen to lipids and DNA and include lipid and DNA hydroperoxides and alkenals arising from the decomposition of lipid hydroperoxides. Studies in the rat and other mammals show the GSH transferases to be dimers in which the subunits are members of a gene super-family. There are three, perhaps four multigene families namely, alpha containing subunits 1, 2, 8 and 10; mu containing subunits 3, 4, 6 and 9; pi containing subunit 7 and subunits 5 and 5* which are so far unassigned. Subunit 5* is apparently restricted to the nucleus and is noteworthy for its activity towards DNA hydroperoxides. Studies in the human are not as advanced as in the rat but so far reveal close similarities. The ability of GSH transferases to detoxify electrophiles is important in carcinogenesis at a number of points. They may inhibit initiation and tumour proportion, but they may be advantageous to the developing tumour cell, and may be acquired in increased amounts during malignant progression. In many tumour cells the development of lines resistant to anticancer drugs is associated with an increased expression of GSH transferases, particularly GSH transferase pi in human cells.     

Effect of cigarette smoke on the mutagenic activation of environmental carcinogens by rodent liver.

In order to assess the effect of cigarette smoke (CS) on metabolic enzymes, male hamsters and rats were exposed for two weeks to smoke produced in a Hamburg type II smoking machine. The livers were then used for Ames liquid incubation and western immunoblot assays. Mutagenic activities of seven heterocyclic amines (HCAs) in Salmonella typhimurium TA98 in the presence of rat or hamster liver S9 were elevated up to 3.7 times above controls (including sham smoke control). Enhancement of mutagenic activities of PhIP and aflatoxin B(1) was observed only in CS-exposed hamster, whereas no significant alteration of mutagenicity was observed with 2-aminofluorene, benzo[a]pyrene, and 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene in strain TA98 or with six N-nitrosodialkylamines in strain TA100. 7,8-Benzoflavone and/or furafylline considerably inhibited the mutagenic activation of IQ and Trp-P-1 in the presence of liver S9 from untreated hamsters and sham smoke- or CS-exposed hamsters and rats, indicating the predominant involvement of hamster cytochrome P450 (CYP) 1A enzymes in the metabolic activation of HCAs. In addition, the data suggest that CS-exposure may selectively induce hepatic CYP1A1/1A2 isoforms. Western immunoblot analyses of liver microsomes using anti-rat CYP antibodies revealed that CS-exposure increased the levels of hamster CYP1A2 (3.9-fold) and rat CYP1A2 (3.0-fold) and CYP1A1, without significant change in the levels of CYP2E1 and CYP2B and 3A isoforms in each species. The presently observed selective induction of HCA activation and CYP isozymes due to CS supports the idea that CS may contribute to enhancing effects on initiation by carcinogens which are metabolically activated by hepatic CYP1A1/1A2. In conjunction with results observed for smokers, the present findings indicate that the hamster is a good animal for studies with CS, and that cigarette smoking in combination with intake of heating protein-rich foods as a life style may markedly contribute to the human carcinogenesis by HCAs.     

Blue cotton, Blue Rayon and Blue Chitin in the analysis of heterocyclic aromatic amines--a review

Heterocyclic amines (HCAs) are a group of compounds formed when protein-rich foods, such as meat or fish, are prepared under normal cooking conditions, such as frying, grilling, or broiling. To evaluate and estimate the risks associated with HCAs contained in the diet, it is important to determine the levels in cooked foods, and the levels of HCAs and metabolites in the body. HCAs are normally found at low amounts in a complex matrix, which necessitates a good purification method and a sensitive detection system. The objective of this review was to briefly present the current knowledge on the use of Blue Cotton, Blue Rayon and Blue Chitin in the analysis of HCAs.     

The role of human glutathione S-transferases (hGSTs) in the detoxification of the food-derived carcinogen metabolite N-acetoxy-PhIP, and the effect of a polymorphism in hGSTA1 on colorectal cancer risk

Food-derived heterocyclic amines (HCAs), particularly 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are implicated in the etiology of human colorectal cancer (CRC) via a process of N-oxidation followed by O-acetylation or O-sulfation to form electrophilic metabolites that react with DNA. Glutathione S-transferases (GSTs) detoxify activated carcinogen metabolites by catalysis of their reaction with GSH. However, among HCAs, only N-acetoxy-PhIP has been shown to be a substrate for the GSTs. By using a competitive DNA-binding assay, we confirm that hGSTA1-1 is an efficient catalyst of the detoxification of N-acetoxy-PhIP. Further, we show that hGSTs A2-2, P1-1, M1-1, T1-1 and T2-2 appear to have low activity towards N-acetoxy-PhIP, and that hGSTs A4-4, M2-2, M4-4 and Z1-1 appear to have no activity towards N-acetoxy-PhIP. A genetic polymorphism in the 5'-regulatory sequence of hGSTA1 has been shown to correlate with the relative and absolute levels of expression of GSTA1/GSTA2 in human liver. Examination of hGSTA1 allele frequency in 100 Caucasian CRC patients and 226 Caucasian controls demonstrated a significant over-representation of the homozygous hGSTA1*B genotype among cases compared to controls (24.0 and 13.7%, respectively, P=0.04). This corresponds to an odds ratio for risk of CRC of 2.0 (95% CI 1.0-3.7) when comparing homozygous hGSTA1*B individuals with all other genotypes. Thus, individuals who are homozygous hGSTA1*B, and who would be predicted to have the lowest levels of hGSTA1 expression in their livers, appear to be at risk of developing CRC, possibly as a result of inefficient hepatic detoxification of N-acetoxy-PhIP.     

DNA Lesions Induced by Replication Stress Trigger Mitotic Aberration and Tetraploidy Development

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.     

Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is associated with tumor development in patients suffering from Chinese herbs nephropathy (now termed aristolochic acid nephropathy, AAN) and may also be a cause for the development of a similar type of nephropathy, the Balkan endemic nephropathy (BEN). Major DNA adducts [7-(deoxyadenosin-N6-yl)-aristolactam and 7-(deoxyguanosin-N2-yl)aristolactam] formed from AA after reductive metabolic activation were found in renal tissues of patients with both diseases. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. This paper reviews major hepatic and renal enzymes responsible for AA-DNA adduct formation in humans. Phase I biotransformation enzymes play a crucial role in the metabolic activation of AA to species forming DNA adducts, while a role of phase II enzymes in this process is questionable. Most of the activation of AA in human hepatic microsomes is mediated by cytochrome P450 (CYP) 1A2 and, to a lower extent, by CYP1A1; NADPH:CYP reductase plays a minor role. In human renal microsomes NADPH:CYP reductase is more effective in AA activation. Prostaglandin H synthase (cyclooxygenase, COX) is another enzyme activating AA in human renal microsomes. Among the cytosolic reductases, NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient in the activation of AA in human liver and kidney. Studies with purified enzymes confirmed the importance of CYPs, NADPH:CYP reductase, COX and NQO1 in the AA activation. The orientation of AA in the active sites of human CYP1A1, -1A2 and NQO1 was predicted from molecular modeling and explains the strong reductive potential of these enzymes for AA detected experimentally. We hypothesized that inter-individual variations in expressions and activities of enzymes activating AA may be one of the causes responsible for the different susceptibilities to this carcinogen reflected in the development of AA-induced nephropathies and associated urothelial cancer.     

Factors determining dietary intakes of heterocyclic amines in cooked foods.

The identification of heterocyclic amines (HCAs) in cooked foods has focused attention on the potential health effects from their consumption in the diet. Recent studies have estimated daily dietary intakes of HCAs that vary 10-fold and implicated different cooked meats as the prime source of HCAs in the diet. These varied estimates can be attributed to the different dietary assessment methods used in these studies, as well as the different levels of HCAs ascribed to the most commonly consumed cooked meats. Epidemiological studies utilizing information on dietary practice and food intake have found higher risks for several cancers among individuals consuming the highest levels of HCAs. These studies have highlighted the importance of using information on cooking methods in addition to food intake to accurately estimate dietary exposure to HCAs.     

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase -- (32)P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.     

DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.     

DNA Damage Response as an Anti-Cancer Barrier: Damage Threshold and the Concept of 'Conditional Haploinsufficiency'

DNA damage response (DDR) emerges as a biological tumorigenesis barrier in early stages of cancer development, and a selective pressure that favors outgrowth of malignant clones with defects in the genome maintenance machinery, such as mutations of p53 and other DDR components. Recent studies indicate that the DDR barrier is not alarmed universally among early noninvasive lesions, but rather responds to high-risk tumorigenic threats that occur in high-grade, pre-malignant lesions that are generally more likely to develop into bona fide malignancies. In addition, while the DDR barrier appears to operate in major types of cancer, such as carcinomas of the lung, breast and colon, DDR activation is rare at any stage of progression among testicular germ-cell tumors. Together with observations that several, but not all oncogenic insults are capable of activating the DDR machinery, these new results point to existence of a critical threshold of such oncogene-induced DNA damage. It seems that only cells and lesions that experience DNA replication stress and DNA damage above such threshold activate the cellular senescence or cell death pathways within the DDR machinery. The higher load of DNA damage may also contribute to cancer predisposition in families with inherited heterozygous defects in the DDR barrier, such as in ATM, BRCA1, BRCA2, p53 and other genes. We propose that carriers of such DDR defects may be more prone to malignancy due to ‘conditional haploinsufficiency’: such partial defects may be asymptomatic in normal tissues, yet they may become manifest under conditions of supra-threshold endogenous DNA damage in oncogene-driven pre-malignant lesions.