Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.     

Development of an O(6)-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O(6)-benzylguanine to the protein

Although it is known that (i) O⁶-alkylguanine-DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O⁶-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-³H]O⁶-benzylguanine ([³H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [³H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [³H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M⁻¹ s⁻¹, respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.     

Topologies of Complexes Containing O-Alkylguanine-DNA Alkyltransferase and DNA

The mutagenic and cytotoxic effects of many alkylating agents are reduced by O⁶-alkylguanine-DNA alkyltransferase (AGT). In humans, this protein not only protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multiprotein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a three-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n + 3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein cross-linking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays, and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions.     

6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O⁶-alkylguanine DNA alkyltransferase

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ∼40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC₅₀ ∼ 76 μM) and repair of DNA containing an O⁶-methylguanine residue (IC₅₀ ∼ 63 μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.     

The role of human O(6)-alkylguanine-DNA alkyltransferase in promoting 1,2-dibromoethane-induced genotoxicity in Escherichia coli

The expression of the DNA repair protein human O(6)-alkylguanine-DNA alkyltransferase (AGT) in Escherichia coli strains GWR109 or TRG8 that lack endogenous AGT greatly increased the toxicity and mutagenicity of 1,2-dibromoethane (DBE). Pretreatment of strain TRG8 expressing human AGT, which is permeable to exogenous drugs, with the AGT inhibitor O(6)-benzylguanine (BG) abolished the lethal and mutagenic effects of DBE, indicating that an active AGT is required for promoting DBE genotoxicity. This was confirmed by the observation that E. coli expressing either the C145A AGT mutant, which is inactive due to loss of the alkyl acceptor site, or mutants Y114E and R128A, which are inactive due to alteration of the DNA binding domain, did not enhance the action of DBE. However, the AGT mutant protein P138M/V139L/P140K, which is active in repairing methylated DNA but is totally resistant to inactivation by BG due to alterations in the active site pocket, was unable to enhance the genotoxicity of DBE. Similarly, other mutants, G156P, Y158H and K165R that are strongly resistant to BG, were much less effective than wild type AGT in mediating the genotoxicity of DBE. Mutant P140A, which is moderately resistant to BG, did increase mutations in response to DBE but was less active than wild type. These results suggest that human AGT is able to interact with a DNA lesion produced by DBE but, instead of repairing it, converts it to a more genotoxic adduct. This interaction is prevented by mutations that modify the active site of AGT to exclude BG.     

Activation of dihaloalkanes by thiol-dependent mechanisms

Dihaloalkanes constitute an important group of chemicals because of their widespread use in industry and agriculture and their potential for causing toxicity and cancer. Chronic toxic effects are considered to depend upon bioactivation, either by oxidation or thiol conjugation. Considerable evidence links genotoxicity and cancer with glutathione conjugations reactions, and some aspects of the mechanisms have been clarified with 1,2-dihaloalkanes and dihalomethanes. Recently the DNA repair protein O6-alkylguanine transferase has been shown to produce cytotoxicity and genotoxicity by means of a thiol-dependent process with similarities to the glutathione reactions.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, IressaR) and benzo[a]pyrene in human lung cancer

Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) affects MAPKs transduction pathways. Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[a]P co-treatment. Gefitinib enhances B[a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[a]P- and gefitinib-induced cytotoxicity, and B[a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.     

Paradoxical enhancement of the toxicity of 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase

The presence of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) paradoxically increases the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli. This enhancement of genotoxicity did not occur when the inactive C145A mutant of human AGT (hAGT) was used. Also, hAGT did not enhance the genotoxicity of S-(2-haloethyl)glutathiones that mimic the reactive product of the reaction of DBE with glutathione, which is catalyzed by glutathione S-transferase. These experiments support a mechanism by which hAGT activates DBE. Studies in vitro showed a direct reaction between purified recombinant hAGT and DBE resulting in a loss of AGT repair activity and a formation of an hAGT-DBE conjugate at Cys(145). A 2-hydroxyethyl adduct was found by mass spectrometry to be present in the Gly(136)-Arg(147) peptide from tryptic digests of AGT reacted with DBE. Incubation of AGT with DBE and oligodeoxyribonucleotides led to the formation of covalent AGT-oligonucleotide complexes. These results indicate that DBE reacts at the active site of AGT to generate an S-(2-bromoethyl) intermediate, which forms a highly reactive half-mustard at Cys(145). In the presence of DNA, the DNA-binding function of AGT facilitates formation of DNA adducts. In the absence of DNA, the intermediate undergoes hydrolytic decomposition to form AGT-Cys(145)-SCH(2)CH(2)OH.     

Stevioside

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.     

Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity

N-Nitroso compounds, such as N-nitrosodiethylamine (NDEA), are a versatile group of chemical carcinogens, being suspected to be involved in gastrointestinal tumors in humans. The intestinal microflora can modify a wide range of environmental chemicals either directly or in the course of enterohepatic circulation. Nitroreductases from bacteria seem to have a wide spectrum of substrates, as observed by the reduction of several nitroaromatic compounds, but their capacity to metabolize N-nitroso compounds has not been described. To elucidate the participation of nitroreductase or acetyltransferase enzymes in the mutagenic activity of NDEA, the bacterial (reverse) mutation test was carried out with the strains YG1021 (nitroreductase overexpression), YG1024 (acetyltransferase overexpression), TA98NR (nitroreductase deficient), and TA98DNP6 (acetyltrasferase deficient), and YG1041, which overexpresses both enzymes. The presence of high levels of acetyltransferase may generate toxic compounds that must be eliminated by cellular processes or can lead to cell death, and consequently decrease the mutagenic effect, as can be observed by the comparison of strain TA98DNP6 with the strains TA98 and YG1024. The slope curves for TA98 strain were 0.66 rev/microM (R(2) = 0.51) and 52.8 rev/microM (R(2) = 0.88), in the absence and presence of S9 mix, respectively. For YG1024 strain, the slope curve, in the presence of S9 mix was 6897 rev/microM (R(2) = 0.78). Our data suggest that N-nitroso compounds need to be initially metabolized by enzymes such as cytochromes P450 to induce mutagenicity. Nitroreductase stimulates toxicity, while acetyltransferase stimulates mutagenicity, and nitroreductase can neutralize the mechanism of mutagenicity generating innoccuos compounds, probably by acting on the product generated after NDEA activation.     

The C-terminal residues of the 2b protein of Cucumber mosaic virus are important for efficient expression in Escherichia coli and DNA-binding

The RNA silencing suppressor 2b protein of Cucumber mosaic virus (CMV) is difficult to produce in Escherichia coli. We compared two CMV 2b proteins that differ in their toxicity against E. coli and found that the acidic amino acid residues in the C-terminal significantly affected the toxicity and expression level of the protein in E. coli. In addition, in a DNA-binding assay, 2b had the ability to bind to DNA, and this ability was affected by the charge on the C-terminal residues of 2b. We concluded that the C-terminal residues were important for 2b’s DNA-binding ability, which may partly explain the toxicity of the protein.     

Antimicrobial and antioxidant activities with acute toxicity,cytotoxicity and mutagenicity of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the coast of Urla (Izmir,Turkey)

The aim of the study was to evaluate the biological activities with toxic properties of the methanol, hexane, and chloroform extracts of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the Coast of Urla in the Aegean Sea. The extracts of C. compressa were tested for their antimicrobial and antioxidant activities in this study. Cytotoxic and mutagenic potentials of the extracts were also evaluated using cell culture and mutagenicity assays. Hexane extract was found to have higher total flavonoid and phenolic contents than the other extracts and exerted higher antioxidant activity than other extracts. All extracts exhibited moderate antimicrobial activity against tested microorganisms (minimum inhibitory concentration ranges are 32–256 μg/mL). The results indicated that the extracts had no significant cytotoxic activity against human hepatocellular carcinoma Hep 3B cell line in all treated concentrations (5–50 μg/mL) and did not show mutagenicity in the Ames test. Lethality was not observed among mice treated with oral doses of the extracts. In conclusion, results of investigations indicate that brown alga C. compressa is a natural source of antioxidant. It has moderate antimicrobial activities with no toxicity.     

Specificity of replicative and SOS-inducible DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains overexpressing SOS-inducible DNA polymerases to 30 chemical mutagens

DNA replication is frequently hindered because of the presence of DNA lesions induced by endogenous and exogenous genotoxic agents. To circumvent the replication block, cells are endowed with multiple specialized DNA polymerases that can bypass a variety of DNA damage. To better understand the specificity of specialized DNA polymerases to bypass lesions, we have constructed a set of derivatives of Salmonella typhimurium TA1538 harboring plasmids carrying the polB, dinB or mucAB genes encoding Escherichia coli DNA polymerase II, DNA polymerase IV or DNA polymerase RI, respectively, and examined the mutability to 30 chemicals. The parent strain TA1538 possesses CGCGCGCG hotspot sequence for -2 frameshift. Interestingly, the chemicals could be classified into four groups based on the mutagenicity to the derivatives: group I whose mutagenicity was highest in strain YG5161 harboring plasmid carrying dinB; group II whose mutagenicity was almost equally high in strain YG5161 and strain TA98 harboring plasmid carrying mucAB; group III whose mutagenicity was highest in strain TA98; group IV whose mutagenicity was not affected by the introduction of any of the plasmids. Introduction of plasmid carrying polB did not enhance the mutagenicity except for benz[a]anthracene. We also introduced a plasmid carrying polA encoding E. coli DNA polymerase I to strain TA1538. Strikingly, the introduction of the plasmid reduced the mutagenicity of chemicals belonging to groups I, II and III, but not the chemicals of group IV, to the levels observed in the derivative whose SOS-inducible DNA polymerases were all deleted. These results suggest that (i) DNA polymerase IV and DNA polymerase RI possess distinct but partly overlapping specificity to bypass lesions leading to -2 frameshift, (ii) the replicative DNA polymerase, i.e., DNA polymerase III, participates in the mutagenesis and (iii) the enhanced expression of E. coli polA may suppress the access of Y-family DNA polymerases to the replication complex.     

Roles of replicative and specialized DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains lacking one or all of SOS-inducible DNA polymerases to 26 chemicals

Progression of DNA replication is occasionally blocked by endogenous and exogenous DNA damage. To circumvent the stalling of DNA replication, cells possess a variety of specialized DNA polymerases that replicate through DNA damage. Salmonella typhimurium strain TA1538 has six DNA polymerases and four of them are encoded by damage-inducible SOS genes, i.e. polB(ST) (pol II), dinB(ST) (pol IV), umuDC(ST) (pol V) and samAB. The strain has been used for the detection of a variety of chemical mutagens because of the high sensitivity to -2 frameshift occurring in CGCGCGCG sequence. To assign the role of each DNA polymerase in the frameshift mutagenesis, we have constructed the derivatives lacking one or all of SOS-inducible DNA polymerases and examined the mutability to 26 chemical mutagens. Interestingly, the chemicals could be categorized into four classes: class I whose mutagenicity was reduced by the deletion of dinB(ST) (1-aminoanthracene and other four chemicals); class II whose mutagenicity was reduced by the deletion of either dinB(ST) or umuDC(ST) plus samAB (7,12-dimethylbenz[a]anthracene and other three chemicals); class III whose mutagenicity largely depended on the presence of umuDC(ST) plus samAB (1-N-6-azabenzo[a]pyrene and other three chemicals) and class IV whose mutagenicity was not reduced by deletion of any of the genes encoding SOS-inducible DNA polymerases (Glu-P-1 and other 12 chemicals). Deletion of polB(ST) reduced by 30-60% the mutagenicity of six chemicals of classes II and III. These results suggest that multiple DNA polymerases including the replicative DNA polymerase, i.e. DNA polymerase III holoenzyme, play important roles in chemically induced -2 frameshift and also that different sets of DNA polymerases are engaged in the translesion bypass of different DNA lesions.     

Insight into the cooperative DNA binding of the O6-alkylguanine DNA alkyltransferase

The O⁶-alkylguanine DNA alkyltransferase (AGT) is a highly conserved protein responsible for direct repair of alkylated guanine and to a lesser degree thymine bases. While specific DNA lesion-bound complexes in crystal structures consist of monomeric AGT, several solution studies have suggested that cooperative DNA binding plays a role in the physiological activities of AGT. Cooperative AGT–DNA complexes have been described by theoretical models, which can be tested by atomic force microscopy (AFM). Direct access to structural features of AGT–DNA complexes at the single molecule level by AFM imaging revealed non-specifically bound, cooperative complexes with limited cluster length. Implications of cooperative binding in AGT–DNA interactions are discussed.     

Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment

Aromatic amines represent one of the most important classes of industrial and environmental chemicals: many of them have been reported to be powerful carcinogens and mutagens, and/or hemotoxicants. Their toxicity has been studied also with quantitative structure-activity relationship (QSAR) methods: these studies are potentially suitable for investigating mechanisms of action and for estimating the toxicity of compounds lacking experimental determinations. In this paper, we first summarized the QSAR models for the rodent carcinogenicity of the aromatic amines. The gradation of potency of the carcinogenic amines depended firstly on their hydrophobicity, and secondly on electronic (reactivity, propensity to be metabolically transformed) and steric properties. On the contrary, the difference between carcinogenic and non-carcinogenic aromatic amines depended mainly on electronic and steric properties. These QSARs can be used directly for estimating the carcinogenicity of aromatic amines. A two-step prediction is possible: (1) estimation of yes/no activity; (2) if the answer from step 1 is yes, then prediction of the degree of potency. The QSARs for rodent carcinogenicity were put in a wider context by comparing them with those for: (a) Salmonella mutagenicity; (b) general toxicity; (c) enzymatic reactions; (d) physical-chemical reactions. This comparative QSAR exercise generated a coherent global picture of the action mechanisms of the aromatic amines. The QSARs for carcinogenicity were similar to those for Salmonella mutagenicity, thus pointing to a similar mechanism of action. On the contrary, the general toxicity QSARs (both in vitro and in vivo systems) were mostly based on hydrophobicity, pointing to an aspecific mechanism of action much simpler than that for carcinogenicity and mutagenicity. The oxidation of the amines (first step in the main metabolic pathway leading to carcinogenic and mutagenic species) had identical QSARs in both enzymatic and physical-chemical systems, thus providing evidence for the link between simple chemical reactions and those in biological systems. The results show that it is possible to generate mechanistically and statistically sound QSAR models for rodent carcinogenicity, and indirectly that the rodent bioassay is a reliable source of good quality data.     

Algicidal and antifungal compounds from the roots of Ruta graveolens and synthesis of their analogs

Bioassay-guided fractionation of the ethyl acetate extract of Ruta graveolens roots yielded rutacridone epoxide with potent selective algicidal activity towards the 2-methyl-isoborneol (MIB)-producing blue-green alga Oscillatoria perornata, with relatively little effect on the green alga Selenastrum capricornutum. The diol-analog of rutacridone epoxide, gravacridondiol, which was also present in the same extract, had significantly less activity towards O. perornata. Rutacridone epoxide also showed significantly higher activity than commercial fungicides captan and benomyl in our micro-bioassay against the agriculturally important pathogenic fungi Colletotrichum fragariae, C. gloeosporioides, C. acutatum, and Botrytis cineara and Fusarium oxysporium. Rutacridone epoxide is reported as a direct-acting mutagen, precluding its use as an agrochemical. In order to understand the structure-activity relationships and to develop new potential biocides without toxicity and mutagenicity, some analogs containing the (2-methyloxiranyl)-dihydrobenzofuran moiety with an epoxide were synthesized and tested. None of the synthetic analogs showed comparable activities to rutacridone epoxide. The absolute stereochemistry of rutacridone was determined to be 2'(R) and that of rutacridone epoxide to be 2'(R), 3'(R) by CD and NMR analysis.