Structural basis of Qng1-mediated salvage of the micronutrient queuine from queuosine-5'-monophosphate as the biological substrate.

Eukaryotic life benefits from-and ofttimes critically relies upon-the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5'-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.

Knockout of ABCC1 in NCI-H441 cells reveals CF to be a suboptimal substrate to study MRP1 activity in organotypic in vitro models.

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an efflux transporter responsible for the extrusion of endogenous substances as well as xenobiotics and their respective metabolites. Its high expression levels in lung tissue imply a key role in pulmonary drug disposition. Moreover, its association with inflammatory lung diseases underline MRP1's relevance in drug development and precision-medicine. With the aim to develop a tool to better understand MRP1's role in drug disposition and lung disease, we generated an ABCC1-/- clone based on the human distal lung epithelial cell line NCI-H441 via a targeted CRISPR/Cas9 approach. Successful knockout (KO) of MRP1 was confirmed by qPCR, immunoblot and Sanger sequencing. To assess potential compensatory upregulation of transporters with a similar substrate recognition pattern as MRP1, expression levels of MRP2-9 as well as OAT1-4, 6, 7 and 10 were measured. Functional transporter activity was determined via release studies with two prodrug/substrate pairs, i.e. 5(6)-carboxyfluorescein (CF; formed from its diacetate prodrug) and S-(6-(7-methylpurinyl))glutathione (MPG; formed from its prodrug 6-bromo-7-methylpurine, BMP), respectively. Lastly, transepithelial electrical resistance (TEER) of monolayers of the KO clone were compared with wildtype (WT) NCI-H441 cells. Of eight initially generated clones, the M2 titled clone showed complete absence of mRNA and protein in accordance with the designed genome edit. In transport studies using the substrate CF, however, no differences between the KO clone and WT NCI-H441 cells were observed, whilst no differences in expression of potential compensatory transporters was noted. On the other hand, when using BMP/MPG, the release of MPG was reduced to 11.5% in the KO clone. Based on these results, CF appears to be a suboptimal probe for the study of MRP1 function, particularly in organotypic in vitro and ex vivo models. Our ABCC1-/- NCI-H441 clone further retained the ability to form electrically tight barriers, making it a useful model to study MRP1 function in vitro.

Design and synthesis of multifunctional microtubule targeting agents endowed with dual pro-apoptotic and anti-autophagic efficacy.

Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.

CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages.

SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1-/- mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.

A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds.

CD44 is emerging as an important receptor biomarker for various cancers. Amongst these is oral cancer, where surgical resection remains an essential mode of treatment. Unfortunately, surgery is frequently associated with permanent disfigurement, malnutrition, and functional comorbidities due to the difficultly of tumour removal. Optical imaging agents that can guide tumour tissue identification represent an attractive approach to minimising the impact of surgery. Here, we report the synthesis of a water-soluble fluorescent probe, namely HA-FA-HEG-OE (compound 1), that comprises components originating from natural sources: oleic acid, ferulic acid and hyaluronic acid. Compound 1 was found to be non-toxic, displayed aggregation induced emission and accumulated intracellularly in vesicles in SCC-9 oral squamous cells. The uptake of 1 was fully reversible over time. Internalization of compound 1 occurs through receptor mediated endocytosis; uniquely mediated through the CD44 receptor. Uptake is related to tumorigenic potential, with non-tumorigenic, dysplastic DOK cells and poorly tumorigenic MCF-7 cells showing only low intracellular levels and highlighting the critical role of endocytosis in cancer progression and metastasis. Together, the recognised importance of CD44 as a cancer stem cell marker in oral cancer, and the reversible, non-toxic nature of 1, makes it a promising agent for real time intraoperative imaging.

Spiroindoline-Capped Selective HDAC6 Inhibitors: Design, Synthesis, Structural Analysis, and Biological Evaluation.

Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Queuine Micronutrient Deficiency Promotes Warburg Metabolism and Reversal of the Mitochondrial ATP Synthase in Hela Cells.

Queuine is a eukaryotic micronutrient, derived exclusively from eubacteria. It is incorporated into both cytosolic and mitochondrial transfer RNA to generate a queuosine nucleotide at position 34 of the anticodon loop. The transfer RNA of primary tumors has been shown to be hypomodified with respect to queuosine, with decreased levels correlating with disease progression and poor patient survival. Here, we assess the impact of queuine deficiency on mitochondrial bioenergetics and substrate metabolism in HeLa cells. Queuine depletion is shown to promote a Warburg type metabolism, characterized by increased aerobic glycolysis and glutaminolysis, concomitant with increased ammonia and lactate production and elevated levels of lactate dehydrogenase activity but in the absence of significant changes to proliferation. In intact cells, queuine deficiency caused an increased rate of mitochondrial proton leak and a decreased rate of ATP synthesis, correlating with an observed reduction in cellular ATP levels. Data from permeabilized cells demonstrated that the activity of individual complexes of the mitochondrial electron transport chain were not affected by the micronutrient. Notably, in queuine free cells that had been adapted to grow in galactose medium, the re-introduction of glucose permitted the mitochondrial F1FO-ATP synthase to operate in the reverse direction, acting to hyperpolarize the mitochondrial membrane potential; a commonly observed but poorly understood cancer trait. Together, our data suggest that queuosine hypomodification is a deliberate and advantageous adaptation of cancer cells to facilitate the metabolic switch between oxidative phosphorylation and aerobic glycolysis.

Malonylation of GAPDH is an inflammatory signal in macrophages.

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.

The queuine micronutrient: charting a course from microbe to man.

Micronutrients from the diet and gut microbiota are essential to human health and wellbeing. Arguably, among the most intriguing and enigmatic of these micronutrients is queuine, an elaborate 7-deazaguanine derivative made exclusively by eubacteria and salvaged by animal, plant and fungal species. In eubacteria and eukaryotes, queuine is found as the sugar nucleotide queuosine within the anticodon loop of transfer RNA isoacceptors for the amino acids tyrosine, asparagine, aspartic acid and histidine. The physiological requirement for the ancient queuine molecule and queuosine modified transfer RNA has been the subject of varied scientific interrogations for over four decades, establishing relationships to development, proliferation, metabolism, cancer, and tyrosine biosynthesis in eukaryotes and to invasion and proliferation in pathogenic bacteria, in addition to ribosomal frameshifting in viruses. These varied effects may be rationalized by an important, if ill-defined, contribution to protein translation or may manifest from other presently unidentified mechanisms. This article will examine the current understanding of queuine uptake, tRNA incorporation and salvage by eukaryotic organisms and consider some of the physiological consequence arising from deficiency in this elusive and lesser-recognized micronutrient.

Queuosine deficiency in eukaryotes compromises tyrosine production through increased tetrahydrobiopterin oxidation.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.

Nrf2 and selenoproteins are essential for maintaining oxidative homeostasis in erythrocytes and protecting against hemolytic anemia.

Reactive oxygen species (ROS) are highly destructive toward cellular macromolecules. However, moderate levels of ROS can contribute to normal cellular processes including signaling. Herein we evaluate the consequence of a pro-oxidant environment on hematopoietic homeostasis. The NF-E2 related factor 2 (Nrf2) transcription factor regulates genes related to ROS scavenging and detoxification. Nrf2 responds to altered cellular redox status, such as occurs with loss of antioxidant selenoproteins after deletion of the selenocysteine-tRNA gene (Trsp). Conditional knockout of the Trsp gene using Mx1-inducible Cre-recombinase leads to selenoprotein deficiency and anemia on a wild-type background, whereas Trsp:Nrf2 double deficiency dramatically exacerbates the anemia and increases intracellular hydrogen peroxide levels in erythroblasts. Results indicate that Nrf2 compensates for defective ROS scavenging when selenoproteins are lost from erythroid cells. We also observed thymus atrophy in single Trsp-conditional knockout mice, suggesting a requirement for selenoprotein function in T-cell differentiation within the thymus. Surprisingly, no changes were observed in the myelomonocytic or megakaryocytic populations. Therefore, our results show that selenoprotein activity and the Nrf2 gene battery are particularly important for oxidative homeostasis in erythrocytes and for the prevention of hemolytic anemia.

Deletion of the selenocysteine tRNA gene in macrophages and liver results in compensatory gene induction of cytoprotective enzymes by Nrf2.

The selenocysteine tRNA (tRNA(Sec)) molecule is the sight of synthesis for the amino acid selenocysteine and the adaptor for its translational insertion into selenoprotein enzymes, the majority of which contribute to cellular redox homeostasis. To examine the consequences of selenoprotein depletion on the oxidative environment of the cell, we generated a conditional knock-out mouse for the tRNA(Sec) gene (Trsp). Deletion of Trsp in either macrophages or liver elevated oxidative stress and activated the transcriptional induction of cytoprotective antioxidant and detoxification enzyme genes, including glutathione S-transferase P1 and NAD(P)H:quinone oxidoreductase 1, and other well known target genes of the transcription factor Nrf2 (NF-E2-related factor 2). Simultaneous disruption of Trsp and Nrf2 severely compromised the cytoprotective response. Double knock-out macrophages displayed reduced viability, elevated oxidative stress, and increased susceptible to hydrogen peroxide treatment compared with deletion of either gene alone. Mice carrying a liver-specific deletion of Trsp on an Nrf2-null background experienced hepatocellular apoptosis and displayed a severely reduced survival rate compared with loss of Trsp alone. Our results thus demonstrate that reduced selenoprotein activity is counterbalanced by an Nrf2-mediated cytoprotective response, which is essential for maintaining cellular redox homeostasis and viability.

The study using wild-type and Ogg1 knockout mice exposed to potassium bromate shows no tumor induction despite an extensive accumulation of 8-hydroxyguanine in kidney DNA.

In order to assess the effect of potassium bromate (KBrO3) on the induction of tumor formation, a 1-year carcinogenesis study was performed using Ogg1 knockout mice (Ogg1(-/-)) and wild-type mice (Ogg1(+/+)). The mice were chronically exposed to KBrO3 by putting it in the drinking water for 29 weeks, at 2 g/l for the first 18 weeks, and then at 1 g/l for another 11 weeks. After termination of treatment the mice were kept for an additional 23 weeks. The amount of 8-hydroxydeoxyguanosine (8-OH-dG) in kidney DNA after 29 weeks of KBrO3 exposure reached 500 8-OH-dG/10(6) dG, almost 250-fold that of untreated wild-type mice. During the course of study the mice appeared normal, although a decrease of body weight gain in both Ogg1(-/-) and Ogg1(+/+) mice exposed to KBrO3, and some kidney malfunction in KBrO3 treated Ogg1(-/-) mice was observed. Surprisingly, when Ogg1(-/-) and Ogg1(+/+) mice were sacrificed at 52 weeks, no tumor formation could be found in kidney or other organs such as lung, liver, spleen, thymus, stomach and intestine. Microscopic examination also showed the absence of precancerous foci in all tissues of both Ogg1(-/-) and Ogg1(+/+) mice. A possible explanation is presented to reconcile these results with those of others which showed an increased incidence of tumor formation in untreated Ogg1(-/-) mice.

Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase.

Tyrosyl-tRNA synthetase (TyrRS) has been studied extensively by mutational and structural analyses to elucidate its catalytic mechanism. TyrRS has the HIGH and KMSKS motifs that catalyze the amino acid activation with ATP. In the present study, the crystal structures of the Escherichia coli TyrRS catalytic domain, in complexes with l-tyrosine and a l-tyrosyladenylate analogue, Tyr-AMS, were solved at 2.0A and 2.7A resolution, respectively. In the Tyr-AMS-bound structure, the 2'-OH group and adenine ring of the Tyr-AMS are strictly recognized by hydrogen bonds. This manner of hydrogen-bond recognition is conserved among the class I synthetases. Moreover, a comparison between the two structures revealed that the KMSKS loop is rearranged in response to adenine moiety binding and hydrogen-bond formation, and the KMSKS loop adopts the more compact ("semi-open") form, rather than the flexible, open form. The HIGH motif initially recognizes the gamma-phosphate, and then the alpha and gamma-phosphates of ATP, with a slight rearrangement of the residues. The other residues around the substrate also accommodate the Tyr-AMS. This induced-fit form presents a novel "snapshot" of the amino acid activation step in the aminoacylation reaction by TyrRS. The present structures and the T.thermophilus TyrRS ATP-free and bound structures revealed that the extensive induced-fit conformational changes of the KMSKS loop and the local conformational changes within the substrate binding site form the basis for driving the amino acid activation step: the KMSKS loop adopts the open form, transiently shifts to the semi-open conformation according to the adenosyl moiety binding, and finally assumes the rigid ATP-bound, closed form. After the amino acid activation, the KMSKS loop adopts the semi-open form again to accept the CCA end of tRNA for the aminoacyl transfer reaction.

Cell proliferation in liver of Mmh/Ogg1-deficient mice enhances mutation frequency because of the presence of 8-hydroxyguanine in DNA.

The Mmh/Ogg1 gene product maintains the integrity of the genome by removing the damaged base 8-hydroxyguanine (8-OH-G), one of the major DNA lesions generated by reactive oxygen species. Using Ogg1-deficient mice, we sought to establish if cells having high amounts of 8-OH-G have the ability to proliferate and whether the mutation frequency increases after proliferation in vivo. When KBrO(3), a known renal carcinogen, at a dose of 2 grams/liter was administered to Ogg1 mutant mice for 12 weeks, the amount of 8-OH-G in liver DNA from treated Ogg1(-/-) mice increased 26.1 times that of treated Ogg1(+/+) mice. The accumulated 8-OH-G did not decrease 4 weeks after cessation of KBrO(3) treatment. Partial hepatectomy was performed on Ogg1(+/-) and Ogg1(-/-) mice after being treated with KBrO(3) for 12 weeks. The remnant liver from Ogg1(-/-) mice treated with KBrO(3) regenerated to the same extent as nontreated Ogg1(+/-) mice. In addition, 8-OH-G was not repaired during cell proliferation by partial hepatectomy, indicating that there is no replication coupled repair of preexisting 8-OH-G. The mutation frequency after the regeneration of liver from treated Ogg1(-/-) mice showed a 3.5-fold increase compared with before regeneration. This represents a mutation frequency 6.2 times that of normal levels. The proliferation of cells having accumulated amounts of 8-OH-G caused mainly GC-->TA transversions. These results showed that inactivation of the Ogg1 gene leads to a higher risk of cancer because cells with accumulated 8-OH-G still retain the ability to proliferate, leading to an increase in the mutation frequency.

Novel homodimeric and heterodimeric rat gamma-hydroxybutyrate synthases that associate with the Golgi apparatus define a distinct subclass of aldo-keto reductase 7 family proteins.

The aldo-keto reductase (AKR) 7 family is composed of the dimeric aflatoxin B(1) aldehyde reductase (AFAR) isoenzymes. In the rat, two AFAR subunits exist, designated rAFAR1 and rAFAR2. Herein, we report the molecular cloning of rAFAR2, showing that it shares 76% sequence identity with rAFAR1. By contrast with rAFAR1, which comprises 327 amino acids, rAFAR2 contains 367 amino acids. The 40 extra residues in rAFAR2 are located at the N-terminus of the polypeptide as an Arg-rich domain that may form an amphipathic alpha-helical structure. Protein purification and Western blotting have shown that the two AFAR subunits are found in rat liver extracts as both homodimers and as a heterodimer. Reductase activity in rat liver towards 2-carboxybenzaldehyde (CBA) was resolved by anion-exchange chromatography into three peaks containing rAFAR1-1, rAFAR1-2 and rAFAR2-2 dimers. These isoenzymes are functionally distinct; with NADPH as cofactor, rAFAR1-1 has a low K(m) and high activity with CBA, whereas rAFAR2-2 exhibits a low K(m) and high activity towards succinic semialdehyde. These data suggest that rAFAR1-1 is a detoxication enzyme, while rAFAR2-2 serves to synthesize the endogenous neuromodulator gamma-hydroxybutyrate (GHB). Subcellular fractionation of liver extracts showed that rAFAR1-1 was recovered in the cytosol whereas rAFAR2-2 was associated with the Golgi apparatus. The distinct subcellular localization of the rAFAR1 and rAFAR2 subunits was confirmed by immunocytochemistry in H4IIE cells. Association of rAFAR2-2 with the Golgi apparatus presumably facilitates secretion of GHB, and the novel N-terminal domain may either determine the targeting of the enzyme to the Golgi or regulate the secretory process. A murine AKR protein of 367 residues has been identified in expressed sequence tag databases that shares 91% sequence identity with rAFAR2 and contains the Arg-rich extended N-terminus of 40 amino acids. Further bioinformatic evidence is presented that full-length human AKR7A2 is composed of 359 amino acids and also possesses an additional N-terminal domain. On the basis of these observations, we conclude that AKR7 proteins can be divided into two subfamilies, one of which is a Golgi-associated GHB synthase with a unique, previously unrecognized, N-terminal domain that is absent from other AKR proteins.

High accumulation of oxidative DNA damage, 8-hydroxyguanine, in Mmh/Ogg1 deficient mice by chronic oxidative stress.

8-Hydroxyguanine (8-OH-G) is a major pre-mutagenic lesion generated from reactive oxygen species. The Mmh/Ogg1 gene product plays a major role in maintaining genetic integrity by removing 8-OH-G by way of the base excision repair pathway. To investigate how oxidative stress influences the formation of 8-OH-G in Ogg1 mutant mice, a known oxidative agent, potassium bromate (KBrO(3)), was administered at a dose of 2 g/l in the drinking water to Ogg1(+/+), Ogg1(+/-) and Ogg1(-/-) mice for 12 weeks. Apurinic (AP) site lyase activity, measured by the excision of 8-OH-G from synthetic oligonucleotides, remained unchanged in kidney cell extracts isolated from Ogg1 mutant mice when the mice were pre-treated by KBrO(3). The levels of 8-OH-G in kidney DNA tremendously increased in a time-dependent manner following exposure of Ogg1(-/-) mice to KBrO(3). Of particular note, the amount of 8-OH-G in kidney DNA from Ogg1(-/-) mice treated with KBrO(3) was approximately 70 times that of KBrO(3)-treated Ogg1(+/+) mice. The accumulated 8-OH-G did not decrease 4 weeks after discontinuing treatment with KBrO(3). KBrO(3) treatment for 12 weeks gave rise to increased mutation frequencies at the transgenic gpt gene in Ogg1(+/+) mice kidney. Absence of the Ogg1 gene further enhanced the mutation frequency. Sequence data obtained from gpt mutants showed that the accumulated 8-OH-G caused mainly GC-->TA transversion and deletion. Other mutations including GC-->AT transition also showed a tendency to increase. These results indicate that 8-OH-G, produced by chronic exposure to exogenous oxidative stress agents, is not repaired to any significant extent within the overall genome of Ogg1(-/-) mice kidney.