Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

Bujing Yishi tablets alleviate photoreceptor cells death via the P2X7R/CX3CL1/CX3CR1 pathway in Retinitis Pigmentosa rats.

BACKGROUND: Retinitis pigmentosa (RP) refers to a group of progressive photoreceptor degenerative diseases. The activation of microglia has been reported to play an important role in the photoreceptor degeneration in RP retinal. Bujing Yishi tablets (BJYS), a Chinese herbal medicine, has been used to treat retinal diseases in China with desirable effect in improving visual function. However, the mechanisms underlying the efficacy of BJYS treatment in RP are not yet fully understood. PURPOSE: Based on the preliminary experiments, this study aimed to investigate the therapeutic mechanism involved in treating N-Methyl-N-Nitrosourea (MNU)-induced retinal degeneration of RP with BJYS. METHODS: To explore the efficacy of BJYS, a rat experimental RP model was established through intraperitoneal injection of MNU (50 mg/kg). Two experiment was carried out. After the treatment, we conducted H&E, TUNEL, retinal cytokine levels and IBA-1 expression in microglia to confirm the impact on RP model. The specific mechanism of action of BJYS tablet was assessed by western blot, real-time polymerase chain reaction (RT-PCR), and immunofluorescence to determine the mRNA and protein expression levels involved in clarifying the effectiveness of BJYS exerted through P2X7R/CX3CL1/CX3CR1 pathway. RESULTS: Significant alleviation of retinal morphological structure and photoreceptor degeneration by BJYS treatment was observed in the retinal of MNU-induced RP rats, BJYS prevented the reduction of ONL thickness and decreased the level of apoptotic cells in ONL. It also inhibited microglia overactivation and reduced retinal levels of IL-1ß, IL-6, TNF-α. In addition, BJYS decreased the protein expression and mRNA expression of P2X7, CX3CL1 and CX3CR1 and reduced the phosphorylation of p38 MAPK. CONCLUSION: In summary, this study suggested that BJYS treatment could alleviate photoreceptors degeneration of RP by inhibiting microglia overactivation and inflammation through the P2X7R/CX3CL1/CX3CR1 pathway. These effects suggest that BJYS tablets may serve as a promising oral therapeutic agent for RP.

High-throughput detection of craniofacial defects in fluorescent zebrafish.

Losses and malformations of cranial neural crest cell (cNCC) derivatives are a hallmark of several common brain and face malformations. Nevertheless, the etiology of these cNCC defects remains unknown for many cases, suggesting a complex basis involving interactions between genetic and/or environmental factors. However, the sheer number of possible factors (thousands of genes and hundreds of thousands of toxicants) has hindered identification of specific interactions. Here, we develop a high-throughput analysis that will enable faster identification of multifactorial interactions in the genesis of craniofacial defects. Zebrafish embryos expressing a fluorescent marker of cNCCs (fli1:EGFP) were exposed to a pathway inhibitor standard or environmental toxicant, and resulting changes in fluorescence were measured in high-throughput using a fluorescent microplate reader to approximate cNCC losses. Embryos exposed to the environmental Hedgehog pathway inhibitor piperonyl butoxide (PBO), a Hedgehog pathway inhibitor standard, or alcohol (ethanol) exhibited reduced fli1:EGFP fluorescence at one day post fertilization, which corresponded with craniofacial defects at five days post fertilization. Combining PBO and alcohol in a co-exposure paradigm synergistically reduced fluorescence, demonstrating a multifactorial interaction. Using pathway reporter transgenics, we show that the plate reader assay is sensitive at detecting alterations in Hedgehog signaling, a critical regulator of craniofacial development. We go on to demonstrate that this technique readily detects defects in other important cell types, namely neurons. Together, these findings demonstrate this novel in vivo platform can predict developmental abnormalities and multifactorial interactions in high-throughput.

N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement.

The non-heme iron-dependent enzyme SznF catalyzes a critical N-nitrosation step during the N-nitrosourea pharmacophore biosynthesis in streptozotocin. The intramolecular oxidative rearrangement process is known to proceed at the FeII-containing active site in the cupin domain of SznF, but its mechanism has not been elucidated to date. In this study, based on the density functional theory calculations, a unique mechanism was proposed for the N-nitrosation reaction catalyzed by SznF in which a four-electron oxidation process is accomplished through a series of complicated electron transferring between the iron center and substrate to bypass the high-valent FeIV═O species. In the catalytic reaction pathway, the O2 binds to the iron center and attacks on the substrate to form the peroxo bridge intermediate by obtaining two electrons from the substrate exclusively. Then, instead of cleaving the peroxo bridge, the Cε-Nω bond of the substrate is homolytically cleaved first to form a carbocation intermediate, which polarizes the peroxo bridge and promotes its heterolysis. After O-O bond cleavage, the following reaction steps proceed effortlessly so that the N-nitrosation is accomplished without NO exchange among reaction species.

A Peroxodiiron(III/III) Intermediate Mediating Both N-Hydroxylation Steps in Biosynthesis of the N-Nitrosourea Pharmacophore of Streptozotocin by the Multi-domain Metalloenzyme SznF.

The alkylating warhead of the pancreatic cancer drug streptozotocin (SZN) contains an N-nitrosourea moiety constructed from Nω-methyl-l-arginine (l-NMA) by the multi-domain metalloenzyme SznF. The enzyme's central heme-oxygenase-like (HO-like) domain sequentially hydroxylates Nδ and Nω' of l-NMA. Its C-terminal cupin domain then rearranges the triply modified arginine to Nδ-hydroxy-Nω-methyl-Nω-nitroso-l-citrulline, the proposed donor of the functional pharmacophore. Here we show that the HO-like domain of SznF can bind Fe(II) and use it to capture O2, forming a peroxo-Fe2(III/III) intermediate. This intermediate has absorption- and Mössbauer-spectroscopic features similar to those of complexes previously trapped in ferritin-like diiron oxidases and oxygenases (FDOs) and, more recently, the HO-like fatty acid oxidase UndA. The SznF peroxo-Fe2(III/III) complex is an intermediate in both hydroxylation steps, as shown by the concentration-dependent acceleration of its decay upon exposure to either l-NMA or Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA). The Fe2(III/III) cluster produced upon decay of the intermediate has a small Mössbauer quadrupole splitting parameter, implying that, unlike the corresponding product states of many FDOs, it lacks an oxo-bridge. The subsequent decomposition of the product cluster to one or more paramagnetic Fe(III) species over several hours explains why SznF was previously purified and crystallographically characterized without its cofactor. Programmed instability of the oxidized form of the cofactor appears to be a unifying characteristic of the emerging superfamily of HO-like diiron oxidases and oxygenases (HDOs).

An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin.

Small molecules containing the N-nitroso group, such as the bacterial natural product streptozotocin, are prominent carcinogens1,2 and important cancer chemotherapeutics3,4. Despite the considerable importance of this functional group to human health, enzymes dedicated to the assembly of the N-nitroso unit have not been identified. Here we show that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. This biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N-N bond formation. We find that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria-including environmental organisms, plant symbionts and human pathogens-which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites.

Increased degradation rate of nitrososureas in media containing carbonate.

The stability of two nitrosoureas, tauromustine and lomustine, has been investigated in different media and buffers. All media tested, except Leibovitz's L-15 medium, significantly increased the degradation rate of the investigated nitrosoureas at pH 7.4. Sodium bicarbonate seems to be the cause of the observed increase of the degradation rate, since it provides the main buffering capacity of all the media except for Leibovitz's L-15 medium, which is based on phosphate buffer. Other ingredients in the media, such as amino acids, vitamins, and inorganic salts, or the ionic strength of a buffer, did not have any major effect on the degradation rate of the nitrosoureas. These results suggest that media containing carbonated buffer should be avoided when the anti-tumor effect of nitrosoureas is to be investigated in different cell cultures.

Proline-linked nitrosoureas as prolidase-convertible prodrugs in human breast cancer cells.

A number of novel proline-linked nitrosoureas (1-4) were synthesized and examined for cytotoxicity and influence on DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that compound 2, the most active of the series, proved to be only slightly less potent than carmustine. It has also been found that carmustine did not inhibit MCF&-7 cells prolidase activity, while compounds 1-4 significantly increased its activity, when used at 50-250 microM concentrations. Proline-linked nitrosoureas (1-4) also had lower ability to inhibit collagen biosynthesis in MCF-7 cells, compared to carmustine. The expression of beta(1)-integrin receptor and phosphorylated MAPK, ERK(1) and ERK(2) was significantly decreased in MCF-7 cells incubated for 24 h with 60 microM of compounds 2 and 4 compared to the control, untreated cells, whereas under the same conditions carmustine did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the proline-linked nitrosoureas (1-4), represent multifunctional inhibitors of breast cancer cell growth and metabolism.

Structural basis for the adherence of Plasmodium falciparum-infected erythrocytes to chondroitin 4-sulfate and design of novel photoactivable reagents for the identification of parasite adhesive proteins.

A dodecasaccharide motif of the low-sulfated chondroitin 4-sulfate (C4S) mediate the binding of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta. Here we studied the detailed C4S structural requirements by assessing the ability of chemically modified C4S to inhibit IRBC binding to the placental chondroitin sulfate proteoglycan. Replacement of the N-acetyl groups with bulky N-acyl or N-benzoyl substituents had no effect on the inhibitory activity of C4S, whereas reduction of the carboxyl groups abrogated the activity. Dermatan sulfates showed approximately 50% inhibitory activity when compared with C4Ss with similar sulfate contents. These data demonstrate that the C4S carboxyl groups and their equatorial orientation but not the N-acetyl groups are critical for IRBC binding. Conjugation of bulky substituents to the reducing end N-acetylgalactosamine residues of C4S dodecasaccharide had no effect on its inhibitory activity. Based on these results, we prepared photoaffinity reagents for the identification of the parasite proteins involved in C4S binding. Cross-linking of the IRBCs with a radioiodinated photoactivable C4S dodecasaccharide labeled a approximately 22-kDa novel parasite protein, suggesting strongly for the first time that a low molecular weight IRBC surface protein rather than a 200-400-kDa PfEMP1 is involved in C4S binding. Conjugation of biotin to the C4S dodecasaccharide photoaffinity probe afforded a strategy for the isolation of the labeled protein by avidin affinity precipitation, facilitating efforts to identify the C4S-adherent IRBC protein(s). Our results also have broader implications for designing oligosaccharide-based photoaffinity probes for the identification of proteins involved in glycosaminoglycan-dependent attachment of microbes to hosts.

Kinetics of DNA alkylation, depurination and hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of cadmium on DNA alkylation.

Anti benzo[a]pyrene diol epoxide (BPDE) alkylates guanines of DNA at N7 in the major groove and at the exocyclic amino group in the minor groove. In this report we investigated the rates of BPDE hydrolysis, DNA alkylation and subsequent depurination of BPDE-adducted pBR322 DNA fragment using polyacrylamide gel electrophoresis. Preincubation studies showed that it hydrolyzed completely in triethanolamine buffer in <2 min. The depurination kinetics showed that a fraction of the N7 alkylated guanine depurinated rapidly; however a significant amount of N7 guanine alkylation remained stable to spontaneous depurination over a 4-h period. Similar results were obtained for the hydrolysis and alkylation rates of syn isomer but it required nearly 500 times more concentration to induce similar levels of N7 guanine alkylation. Cadmium ion strongly inhibited the N7 guanine alkylation of both isomers. But the minor groove alkylation was not affected as demonstrated by postlabeling assay which confirmed the presence of heat-and cadmium-stable minor groove adducts in BPDE-treated calf thymus DNA. Based on these and our earlier findings, we propose a mechanism for the synergistic effect of cadmium in chemically induced carcinogenesis.

Metabolism of tauromustine in liver and lung microsomes from various species.

1. The cytochrome P450 (CYP)-mediated metabolism of tauromustine has been evaluated in liver and lung microsomes from various species. Liver microsomes from rat pretreated with typical CYP inducers, human liver microsomes and cDNA-expressed human CYP enzymes were used to study the enzymatic basis of the metabolism. The further metabolism of the monodemethylated product of tauromustine and that of the denitrosated product were also investigated. 2. The major routes of tauromustine metabolism were demethylation to the alkylating active compound, R2, and denitrosation to the inactive metabolite, M3. The extent of metabolism and the activity of demethylation versus denitrosation varied among the species. The highest metabolism was found in mouse (BDF strain) followed by dog, rat and the human liver. Tauromustine was also metabolized to a low extent in lung microsomes from these species. 3. The further metabolism of R2 and M3 was approximately 100 times lower in activity than that of tauromustine. Both the demethylation and the denitrosation of tauromustine were increased 3-fold in liver microsomes from rat pretreated with phenobarbital, whereas treatment with cyanopregnenolone enhanced the denitrosation 11-fold, indicating the involvement of CYP3A. 4. Metabolism across a panel of 10 human liver microsomal samples demonstrated a correlation with testosterone 6beta-hydroxylation of demethylation (r2 = 0.86) and denitrosation of tauromustine (r2 = 0.79). Among the human cDNA expressed CYP enzymes, not only was tauromustine determined to be catalysed predominantly by CYP3A4, but also to some extent by CYP2C19 and CYP2D6. 5. In conclusion, the present results indicate a major role of CYP3A enzymes in the metabolism of tauromustine.

Cadmium inhibits BPDE alkylation of DNA in the major groove but not in the minor groove.

Cadmium, a constituent of tobacco, has the potential to act in synergy with other carcinogens in tobacco smoke. Working on the hypothesis that cadmium interactions with DNA enhances the mutagenic lesions induced by tobacco carcinogens, we investigated the site and sequence selectivity of DNA binding by cadmium using DNA reactive chemical probes. Our results show that this divalent cation binds to N7 guanines with a great preference for those occurring in runs of G's. Further, cadmium considerably diminishes N7 guanine alkylation by the tobacco carcinogen metabolite BPDE; however, the biologically relevant guanine alkylation in the minor groove by BPDE was not affected. The relevance of our findings to cadmium's role in the tobacco carcinogenesis is discussed.

Mechanism of action of fotemustine, a new chloroethylnitrosourea anticancer agent: evidence for the formation of two DNA-reactive intermediates contributing to cytotoxicity.

Methyl excision repair deficient human tumor cells (Mer-) were found to be hypersusceptible to killing by the antimelanoma agent fotemustine (FM) implicating alkylation of O6 guanine as the major contributor to toxicity. Preincubation of the drug in aqueous solution for 5 min resulted in an immediate reduction in cytotoxicity (35-50%), in vitro DNA alkylation (31%), and DNA interstrand cross-linking (40%) followed by a second reaction with considerably slower kinetics. Electrospray ionisation mass spectrometry (ESI-MS) showed that in aqueous solution FM rearranged rapidly to form either a metastable tautomer or decomposed to form a highly reactive diazohydroxide (t1/2 < 2 min). These results suggest the presence of two DNA-reactive species relevant to biological activity. Coincubation of ellagic acid (an inhibitor of O6-guanine alkylation) with FM inhibited in vitro ISC, suggesting that the O6-chloroethyl lesion is the predominant cause of the cross-link. On the basis of these findings, we propose that FM breaks down to form a short-lived intermediate, 2-chloroethyldiazohydroxide, which rapidly generates O6-guanine lesions responsible for the drug's initial activity and a long lived iminol tautomer responsible for the remaining O6 guanine alkylation and cytotoxicity.

PCNA: structure, functions and interactions.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This toroidal-shaped protein encircles DNA and can slide bidirectionally along the duplex. One of the well-established functions for PCNA is its role as the processivity factor for DNA polymerase delta and epsilon. PCNA tethers the polymerase catalytic unit to the DNA template for rapid and processive DNA synthesis. In the last several years it has become apparent that PCNA interacts with proteins involved in cell-cycle progression which are not a part of the DNA polymerase apparatus. Some of these interactions have a direct effect on DNA synthesis while the roles of several other interactions are not fully understood. This review summarizes the structural features of PCNA and describes the diverse functions played by the protein in DNA replication and repair as well as its possible role in chromatin assembly and gene transcription. The PCNA interactions with different cellular proteins and the importance of these interactions are also discussed.

[Study of N-nitroso compounds which have NO-release ability].

Nitric oxide (NO), which plays an important role in the vital functions of organisms, is gaseous and labile molecule. Much attention has been paid to the stability and easily handling of NO donors, for careful handling of NO is required during experimental work. We synthesized a series of aromatic N-nitrosoureas and N-nitrosamides which efficiently liberates NO at room temperature. Generation of NO from the aromatic N-nitroso compounds was chemically confirmed by the trapping of NO as a nitrosyl complex of tetraphenylporphyrinatocobalt (II) and spectrophotometrically quantified by means of the Griess reaction using a newly designed test apparatus. 3,3-Dibenzyl-1-(4-tolyl)-1-nitrosourea showed the greatest NO-generating ability among the synthesized N-nitroso compounds. Further, the NO-generating ability was related to the reciprocal of the ID50 value for growth inhibition of cultured L-5178Y cell by the aromatic N-nitroso compounds.

Determination of nitrosourea compounds in brain tissue by gas chromatography and electron capture detection.

A relatively simple, high-sensitivity gas chromatographic assay is described for nitrosourea compounds, such as BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] and MeCCNU [1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea], in small biopsy samples of brain and other tissues. After extraction with ethyl acetate, secondary amines in BCNU and MeCCNU are derivatized with trifluoroacetic anhydride. Compounds are separated and quantitated by gas chromatography using a capillary column with temperature programming and an electron capture detector. Standard curves of BCNU indicate a coefficient of variance of 0.066 +/- 0.018, a correlation coefficient of 0.929, and an extraction efficiency from whole brain of 68% with a minimum detectable amount of 20 ng in 5-10 mg samples. The assay has been facile and sensitive in over 1000 brain biopsy specimens after intravenous and intraarterial infusions of BCNU.

5-Fluorouracil causes alterations in the pharmacokinetic profile of tauromustine in NMRI mice.

The pharmacokinetic profiles of 5-fluorouracil (5-FU) and tauromustine (TCNU) were investigated in NMRI mice following their administration either alone or as part of simultaneous or sequential combinations. The profile of 5-FU remained unaltered irrespective of the sequence of administration. However, following simultaneous administration of 5-FU and TCNU or pretreatment with 5-FU, plasma levels of TCNU were decreased by a factor of 2.5-3 as compared with those seen when TCNU was given alone. These decreased plasma levels were explained by an increase in the TCNU volume of distribution, although this was not due to alterations in TCNU plasma protein binding. These sequence-dependent alterations in the pharmacokinetic profile of TCNU correlate with and may explain previously reported differences in the anti-tumour activity and toxicity profile of this combination.

Measurement and characterization of the denitrosation of tauromustine and related nitrosoureas by glutathione transferases in liver cytosol from various species.

The denitrosation of a novel nitrosourea, 1-(2-chloroethyl)-3-[(2-dimethylaminosulfonyl)-ethyl]-1-nitrosoure a, tauromustine (TCNU), has been investigated in liver cytosol from various species and with a pool of cytosolic rat liver glutathione transferases (GSTs). In addition, the denitrosation of related nitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) have been investigated. By using radioactive nitrosoureas as substrates and HPLC to detect the denitrosated products, a sensitive assay has been developed. The activities towards additional substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and trans-stilbene oxide (tSBO) were also measured in the various cytosol fractions. The apparent Vmax and Km values for the denitrosation of TCNU in rat liver cytosol were calculated to be 830 pmol/min/mg protein and 8.4 mM respectively. The rate of denitrosation of TCNU was found to be highest in the mouse, followed by the rat and the dog. The induction of GSTs by phenobarbital in rats increased the denitrosation of TCNU similar to the activities found towards CDNB and tSBO as substrates. The highest denitrosation activity by GST was determined towards BCNU, followed by CCNU and TCNU. Our results suggest that the denitrosation of various nitrosoureas via GST may play an important role in the deactivating process occurring in vivo and for the efficacy of nitrosoureas used in chemotherapy.

Urinary excretion of O6-butylguanine after the administration of N-nitroso-N-butylurea in rats.

O6-Butylguanine was detected in the urine of rats given the butylating agent N-nitroso-N-butylurea. O6-Butylguanine contents in the 24-h rat urine samples after i.p. doses of 50, 100, and 200 mg/kg N-nitroso-N-butylurea were 1.03 +/- 0.41 (SE), 8.30 +/- 1.70, and 59.53 +/- 6.52 pmol, respectively. This suggests that O6-butylguanine formation in nucleic acids might be repaired in vivo, possibly by base excision, besides other mechanisms. After i.v. doses of 0.1 and 1 mg/kg of O6-butylguanine to rats urinary excretion did not exceed 2% of the administered dose, suggesting that the amount of O6-butylguanine effectively released by base excision might be much larger than that detected in the urine after N-nitroso-N-butylurea. Inhibition of the enzyme O6-alkyl-DNA transferase by N-nitrosodimethylamine increased urinary O6-butylguanine resulting from exposure to N-nitroso-N-butylurea (100 mg/kg i.p.) up to four times, thus confirming an alternative DNA repair mechanism.