Synthesis, Characterization, and Antimicrobial Evaluation of Schiff base-mixed Ligand Complexes with Divalent Metal Ions Derived from Amoxicillin and Vanillin/Nicotinamide.

INTRODUCTION: This study focuses on the development of novel antimicrobial agents. A Schiff base ligand, 6-(2-(4-hydroxy-3-methoxybenzylideneamino)-2-(4-hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo- 4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid, synthesized through the condensation of amoxicillin and vanillin in methanol, served as the foundation. Polydentate mixed ligand complexes were then formed by reacting the Schiff base with metal ions (Fe(II), Co(II), Ni(II), Cu(II), and Zn(II)) and nicotinamide in specific ratios. METHODS: Characterization involved various techniques, such as 1H-NMR, FT-IR, UV-Vis, and elemental analysis for the ligand, and Atomic Absorption, FT-IR, UV-Vis, magnetic susceptibility, and conductance measurements for the Schiff base-metal ion complexes. RESULTS: Quantum chemical features of both ligands and metal complexes were computed, refining their electronic and molecular structures theoretically. Antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Acinetobacter baumannii, and Pseudomonas aeruginosa was assessed for the starting materials, ligands, and synthesized complexes, revealing significant effects on certain species. In-silico binding modes with Escherichia coli (PDB ID: 5iq9) were determined through molecular docking. CONCLUSION: This study underscores the potential applications of the Schiff base ligands and their metal complexes in developing new antimicrobial agents.

Uncovering the Hidden Dangers and Molecular Mechanisms of Excess Folate: A Narrative Review.

This review delves into the intricate relationship between excess folate (vitamin B9) intake, especially its synthetic form, namely, folic acid, and its implications on health and disease. While folate plays a pivotal role in the one-carbon cycle, which is essential for DNA synthesis, repair, and methylation, concerns arise about its excessive intake. The literature underscores potential deleterious effects, such as an increased risk of carcinogenesis; disruption in DNA methylation; and impacts on embryogenesis, pregnancy outcomes, neurodevelopment, and disease risk. Notably, these consequences stretch beyond the immediate effects, potentially influencing future generations through epigenetic reprogramming. The molecular mechanisms underlying these effects were examined, including altered one-carbon metabolism, the accumulation of unmetabolized folic acid, vitamin-B12-dependent mechanisms, altered methylation patterns, and interactions with critical receptors and signaling pathways. Furthermore, differences in the effects and mechanisms mediated by folic acid compared with natural folate are highlighted. Given the widespread folic acid supplementation, it is imperative to further research its optimal intake levels and the molecular pathways impacted by its excessive intake, ensuring the health and well-being of the global population.

High Dietary Folic Acid Intake Is Associated with Genomic Instability in Peripheral Lymphocytes of Healthy Adults.

Mandatory fortification of food with synthetic folic acid (FA) was instituted in 1998 to reduce the incidence of neural tube defects. Adequate folate status is correlated with numerous health benefits. However, elevated consumption of FA is controversially associated with deleterious effects on health. We previously reported that excess FA mimicked folate depletion in a lymphoblastoid cell line. To explore the impact of FA intake from fortified food, we conducted an observational human study on 33 healthy participants aged 18-40 not taking any supplements. Food intake, anthropomorphic measurements, and blood samples were collected and analyzed. Our results show that individuals belonging to the highest tertile of folic acid intake, as well as ones with the highest folic acid to total folate intake ratio (FAR), display a significantly greater incidence of lymphocyte genomic damage. A decrease in global DNA methylation is observed in the highest tertile of FAR compared to the lowest (p = 0.055). A downward trend in the overall gene expression of select DNA repair and one carbon cycle genes (MGMT, MLH1, UNG, MTHFR, MTR) is noted with increased folate status and FA intake. These results provide supporting evidence that high consumption of FA from fortified foods can precipitate genomic instability in peripheral lymphocyte in vivo.

Excessive Folic Acid Mimics Folate Deficiency in Human Lymphocytes.

Food fortification with synthetic folic acid (FA), along with supplementation, results in a marked increase in the population total of serum folates and unmetabolized folic acid (UMFA). Despite the success in reducing neural tube defects at birth in the intended target population (women of childbearing age), the potential deleterious effects of chronically high levels of UMFA in susceptible segments of the population require further investigation. In this study, we examine the effects of FA concentrations, ranging from depletion to supraphysiological levels, on markers of proliferation, DNA methylation, and DNA damage and repair in a human lymphoblastoid cell line (LCL). We note that both low and high levels of FA similarly impact global DNA methylation, cytome biomarkers measured through the CBMN assay, DNA damage induced by oxidative stress, and DNA base excision repair gene expression.

Succinylsulfathiazole modulates the mTOR signaling pathway in the liver of c57BL/6 mice via a folate independent mechanism.

Researchers studying the effect of folate restriction on rodents have resorted to the use of the antibiotic succinylsulfathiazole (SST) in the folate depleted diet to induce a folate deficient status. SST has been used extensively in rodent studies since the 1940s. Its localized effect on the gut bacteria as well as its effectiveness in reducing folate producing species is well documented. The possible overlap between the pathways affected by folate depletion and SST could potentially produce a confounding variable in such studies. In our novel study, we analyzed the effect of SST on folate levels in c57Bl/6 male mice fed folate supplemented and deficient diets. We did not observe any significant difference on growth and weight gain at 21 weeks. SST did not significantly affect folate levels in the plasma, liver and colon tissues; however, it did alter energy metabolism and expression of key genes in the mTOR signaling pathway in the liver. This research sheds light on a possible confounding element when using SST to study folate depletion due to the potential overlap with multiple critical pathways such as mTOR. SUMMARY: The antibiotic succinylsulfathiazole (SST) is used to reduce folate producing bacteria in rodent folate depletion studies. SST can modulate critical energy and nutrient sensing pathways converging onto mTOR signaling, and potentially confounding cancer studies.

The Timing and Duration of Folate Restriction Differentially Impacts Colon Carcinogenesis.

Diet plays a crucial role in the development of colorectal cancer (CRC). Of particular importance, folate, present in foods and supplements, is a crucial modulator of CRC risk. The role of folate, and, specifically, the synthetic variant, folic acid, in the primary prevention of CRC has not been fully elucidated. Animal studies varied considerably in the timing, duration, and supplementation of folates, leading to equivocal results. Our work attempts to isolate these variables to ascertain the role of folic acid in CRC initiation, as we previously demonstrated that folate restriction conferred protection against CRC initiation in a ß-pol haploinsufficient mouse model. Here we demonstrated that prior adaptation to folate restriction altered the response to carcinogen exposure in wild-type C57BL/6 mice. Mice adapted to folate restriction for 8 weeks were protected from CRC initiation compared to mice placed on folate restriction for 1 week, irrespective of antibiotic supplementation. Through analyses of mTOR signaling, DNA methyltransferase, and DNA repair, we have identified factors that may play a critical role in the differential responses to folate restriction. Furthermore, the timing and duration of folate restriction altered these pathways differently in the absence of carcinogenic insult. These results represent novel findings, as we were able to show that, in the same model and under controlled conditions, folate restriction produced contrasting results depending on the timing and duration of the intervention.

Evolutionary features of 8K (KDa) silencing suppressor protein of Potato mop-top virus.

The cysteine-rich 8K protein of Potato mop-top virus (PMTV) suppresses host RNA silencing. In this study, evolutionary analysis of 8K sequences of PMTV isolates was studied on the basis of nucleotide and amino acid sequences. Twenty-one positively selected sites were identified in 8K coding regions. Recombination events were found in the 8K of PMTV isolates with a rate of 1.8. Totally 30 haplotypes were detected with haplotype diversity ranging from 0.8 to 1.0 and nucleotide diversity from 7.58 to 13.62. The positions 33 and 30 indicated the highly positive and negative selection (with the highest and the lowest dN-dS values), respectively. Tajima's D test suggested that 8K is evolving with a strong positive selection for worldwide isolates. High frequency of segregating sites was identified along 204 positions of 8K. Moreover, in this study, we used Shannon entropy-based approach to evaluate the variability of each site of nucleotide and corresponding amino acid. Based on Shannon entropy method, 139 and 97 nucleotide sites had the highest entropy value, while 47 and 33 amino acid sites showed the most diversity along 8K sequences. Our findings suggest that 8K as an RNA silencing suppressor evolves rapidly. Taken together, its variability might play a big threat to infect other plants or overcome resistant cultivars.

SIN3 is critical for stress resistance and modulates adult lifespan.

Coordinate control of gene activity is critical for fitness and longevity of an organism. The SIN3 histone deacetylase (HDAC) complex functions as a transcriptional repressor of many genes. SIN3-regulated genes include those that encode proteins affecting multiple aspects of mitochondrial function, such as energy production and stress responsiveness, important for health maintenance. Here we used Drosophila melanogaster as a model organism to examine the role of SIN3 in the regulation of fitness and longevity. Adult flies with RNA interference (RNAi) induced knockdown expression of Sin3A have reduced climbing ability; an activity that likely requires fully functional mitochondria. Additionally, compared to wild type, adult Sin3A knockdown flies were more sensitive to oxidative stress. Interestingly, media supplementation with the antioxidant glutathione largely restored fly tolerance to oxidative stress. Although Sin3A knockdown flies exhibited decreased longevity compared to wild type, no significant changes in expression of many well-categorized aging genes were observed. We found, however, that Sin3A knockdown corresponded to a significant reduction in expression of genes encoding proteins involved in the de novo synthesis of glutathione. Taken together, the data support a model whereby SIN3 regulates a gene expression program required for proper mitochondrial function and effective stress response during adulthood.

Curcumin is an early-acting stage-specific inducer of extended functional longevity in Drosophila.

Larval feeding with curcumin induces an extended health span with significantly increased median and maximum longevities in the adult fly. This phenotype is diet insensitive and shows no additive effect on longevity when combined with an adult dietary restriction (DR) diet, suggesting that curcumin and DR operate via the same or overlapping pathways for this trait. This treatment significantly slows the aging rate so that it is comparable with that of genetically selected long lived animals. The larval treatment also enhances the adult animal's geotactic activity in an additive manner with DR, suggesting that curcumin and DR may use different pathways for different traits. Feeding the drug to adults during only the health span also results in a significantly extended health span with increased median and maximum life span. This extended longevity phenotype is induced only during these stage-specific periods. Feeding adults with the drug over their whole life results in a weakly negative effect on median longevity with no increase in maximum life span. There are no negative effects on reproduction, although larval curcumin feeding increases development time, and also apparently accelerates the normal late-life neuromuscular degeneration seen in the legs. Gene expression data from curcumin-fed larvae shows that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in longevity extension are also affected. These data support the hypothesis that curcumin acts as if it is a DR mimetic nutraceutical. These data also suggest that the search for DR mimetics may be enhanced by the use of stage-specific screening of candidate molecules.

DNA Repair and Cancer Therapy: Targeting APE1/Ref-1 Using Dietary Agents.

Epidemiological studies have demonstrated the cancer protective effects of dietary agents and other natural compounds isolated from fruits, soybeans, and vegetables on neoplasia. Studies have also revealed the potential for these natural products to be combined with chemotherapy or radiotherapy for the more effective treatment of cancer. In this paper we discuss the potential for targeting the DNA base excision repair enzyme APE1/Ref-1 using dietary agents such as soy isoflavones, resveratrol, curcumin, and the vitamins ascorbate and α-tocopherol. We also discuss the potential role of soy isoflavones in sensitizing cancer cells to the effects of radiotherapy. A comprehensive review of the dual nature of APE1/Ref-1 in DNA repair and redox activation of cellular transcription factors, NF-κB and HIF-1α, is also discussed. Further research efforts dedicated to delineating the role of APE1/Ref-1 DNA repair versus redox activity in sensitizing cancer cells to conventional treatment are warranted.

Folate deficiency regulates expression of DNA polymerase ß in response to oxidative stress.

Folate deficiency has been shown to influence carcinogenesis by creating an imbalance in the base excision repair (BER) pathway, affecting BER homeostasis. The inability to mount a BER response to oxidative stress in a folate-deficient environment results in the accumulation of DNA repair intermediates, i.e., DNA strand breaks. Our data indicate that upregulation of ß-pol expression in response to oxidative stress is inhibited by folate deficiency at the level of gene expression. Alteration in the expression of ß-pol in a folate-deficient environment is not due to epigenetic changes in the core promoter of the ß-pol gene, i.e., the CpG islands within the ß-pol promoter remain unmethylated in the presence or absence of folate. However, the promoter analysis studies show a differential binding of regulatory factors to the -36 to -7 region (the folic acid-response region, FARR) within the core promoter of ß-pol. Moreover, we observe a tight correlation between the level of binding of regulatory factors with the FARR and inhibition of ß-pol expression. Based on these findings, we propose that folate deficiency results in an upregulation/stability of negative regulatory factors interacting with FARR, repressing the upregulation of the ß-pol gene in response to oxidative stress.

Folate deficiency provides protection against colon carcinogenesis in DNA polymerase beta haploinsufficient mice.

Aging and DNA polymerase beta deficiency (beta-pol(+/-)) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency (FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in beta-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in beta-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in beta-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD beta-pol haploinsufficient mice.

Oxidative stress alters base excision repair pathway and increases apoptotic response in apurinic/apyrimidinic endonuclease 1/redox factor-1 haploinsufficient mice.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-kappaB, and the major 5'-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 (Apex(+/-)) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-kappaB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex(+/-) mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex(+/-) mice exposed to 2-NP displayed a significant decline in 3'-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER.

Caloric restriction and genomic stability.

Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents while increasing mean and maximum life spans. It has been suggested that CR extends longevity and reduces age-related pathologies by reducing the levels of DNA damage and mutations that accumulate with age. This hypothesis is attractive because the integrity of the genome is essential to a cell/organism and because it is supported by observations that both cancer and immunological defects, which increase significantly with age and are delayed by CR, are associated with changes in DNA damage and/or DNA repair. Over the last three decades, numerous laboratories have examined the effects of CR on the integrity of the genome and the ability of cells to repair DNA. The majority of studies performed indicate that the age-related increase in oxidative damage to DNA is significantly reduced by CR. Early studies suggest that CR reduces DNA damage by enhancing DNA repair. With the advent of genomic technology and our increased understanding of specific repair pathways, CR has been shown to have a significant effect on major DNA repair pathways, such as NER, BER and double-strand break repair.

Haploinsufficiency in DNA polymerase beta increases cancer risk with age and alters mortality rate.

This study uses a base excision repair (BER)-deficient model, the DNA polymerase beta heterozygous mouse, to investigate the effect of BER deficiency on tumorigenicity and aging. Aged beta-pol(+/-) mice express 50% less beta-pol transcripts and protein (P < 0.05) than aged beta-pol(+/+) mice, showing maintenance of the heterozygous state over the life span of the mouse. This reduction in beta-pol expression was not associated with an increase in mutation rate but was associated with a 100% increase in the onset of hypoploidy. Aged beta-pol(+/-) mice exhibited a 6.7-fold increase in developing lymphoma (P < 0.01). Accordingly, 38% of beta-pol(+/-) mice exhibited lymphoid hyperplasia, whereas none of the beta-pol(+/+) exhibited this phenotype. beta-pol(+/-) mice were also more likely to develop adenocarcinoma (2.7-fold increase; P < 0.05) and more likely to develop multiple tumors, as 20% of the beta-pol(+/-) animals died bearing multiple tumors compared with only 5% of the beta-pol(+/+) animals (P < 0.05). In spite of accelerated tumor development, no gross effect of beta-pol heterozygosity was seen with respect to life span. However, the survival curves for the beta-pol(+/+) and beta-pol(+/-) mice are not identical. A maximum likelihood estimation analysis showed a modest but significant (P < 0.05) acceleration of the age-dependent mortality rate in beta-pol(+/-) mice. Thus, the beta-pol(+/-) mouse represents a model in which mortality rate and tumor development are accelerated and provides evidence supporting the role of genomic maintenance in both aging and carcinogenesis.

Age-related loss of the DNA repair response following exposure to oxidative stress.

Young (4- to 6-month-old) and aged (24- to 28-month-old) mice were exposed to 2-nitropropane (2-NP), a DNA oxidizing agent, and the ability to induce DNA polymerase beta (beta-pol) and AP endonuclease (APE) was determined. In contrast to the inducibility of these gene products in response to oxidative damage in young mice, aged mice showed a lack of inducibility of beta-pol and APE. APE protein level and endonuclease activity were both reduced 40% (p<.01) in response to 2-NP. Accordingly, the accumulation of DNA repair intermediates in response to 2-NP differed with age. Young animals accumulated 3'OH-containing DNA strand breaks, whereas the aged animals did not. A role for p53 in the difference in DNA damage response with age is suggested by the observation that the accumulation of p53 protein in response to DNA damage in young animals was absent in the aged animals. Our results are consistent with a reduced ability to process DNA damage with age.

A sensitive biochemical assay for the detection of uracil.

We have developed a sensitive new assay for the detection of uracil in DNA. The assay described here is an adaptation to the highly sensitive aldehydic slot blot (ASB) assay developed by Nakamura et al. (Nakamura et al. 1998: Cancer Res 58:222-225) in which aldehydic DNA lesions (ADLs) are detected through binding of a biotinylated aldehydic reactive probe to DNA. The uracil DNA glycosylase (UDG)-coupled ASB assay uses uracil-DNA glycosylase to generate an abasic site, which is subsequently detected by the ASB methodology. The ability to modify this technique for the detection of uracil has these advantages: small quantities of DNA are required (4 microg of DNA); the assay is adaptable to DNA from both cells and tissues; sensitivity is as good as that achieved by less accessible methodologies, like gas chromatography-mass spectroscopy (GC-MS); DNA strand breaks are not a confounding variable; preexisting aldehydic lesions are blocked through the use of methoxyamine; variation is very low (<3%); radioactive isotopes are not required; and the assay is easy to establish and involves only equipment and reagents that are inexpensive and readily available. This assay is conceivably adaptable to the detection of other DNA base lesions through the use of a variety of DNA glycosylases.

Structure and regulation of the murine reduced folate carrier gene: identification of four noncoding exons and promoters and regulation by dietary folates.

The upstream structure and regulation of the mouse reduced folate carrier (mRFC) gene was characterized. By 5'-rapid amplification of cDNA ends assay and DNA sequencing from mouse tissues and 7-15-day stage embryos, mRFC transcripts with four unique 5' noncoding exons, designated mRFC-a,-b,-c, and -d, were identified mapping over 6300 bp. The 5' noncoding exons were characterized by multiple transcription starts and, for form b, two alternate splice forms. mRFC transcript forms were measured by real-time reverse transcription-PCR in mouse tissues and embryos and in L1210 leukemia and BNL CL.2 liver cell lines. The highest mRFC levels were detected in kidney and brain. mRFC-b and -c were the major transcript forms, with low levels of mRFC-a and -d. The 5'-flanking regions for exons a-d each exhibited promoter activity in reporter gene assays. mRFC transcripts and individual noncoding exons were measured in small intestine and kidney from mice fed folate-deficient or -replete diets. Mice fed the folate-deficient diet exhibited a significant (13.8-fold) increase in total mRFC transcripts and protein in the small intestine, reflecting increases in each of the mRFC-b, -c, and -d forms. Only minor changes in mRFC transcript levels or distributions were detected for kidney. Levels of folate-binding protein alpha were also increased in both small intestine and kidney in folate-deficient mice (91- and 2-fold, respectively). Multidrug resistance-associated proteins 1 and 3 were, likewise, elevated in intestine from folate-deficient mice (53- and 168-fold, respectively); however, there were no significant changes in kidney. Our results document the existence of four unique noncoding exons and promoters for mRFC and demonstrate a facile induction of mRNAs for mRFC and multidrug resistance-associated proteins 1 and 3 in intestine in response to changes in dietary folate intake.

Imbalanced base excision repair in response to folate deficiency is accelerated by polymerase beta haploinsufficiency.

The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.

Apurinic/apyrimidinic endonuclease (APE/REF-1) haploinsufficient mice display tissue-specific differences in DNA polymerase beta-dependent base excision repair.

Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5'-endonuclease activity and 3'-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex(+/-)). Our data indicate that Apex(+/-) mice are indeed APE-haploinsufficient, as exhibited by a 40-50% reduction (p < 0.05) in APE mRNA, protein, and 5'-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase beta and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase beta-dependent BER response to cellular stress.