Attenuation of Polycyclic Aromatic Hydrocarbon (PAH)-Induced Carcinogenesis and Tumorigenesis by Omega-3 Fatty Acids in Mice In Vivo.

Lung cancer is the leading cause of cancer death worldwide. Polycyclic aromatic hydrocarbons (PAHs) are metabolized by the cytochrome P450 (CYP)1A and 1B1 to DNA-reactive metabolites, which could lead to mutations in critical genes, eventually resulting in cancer. Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are beneficial against cancers. In this investigation, we elucidated the mechanisms by which omega-3 fatty acids EPA and DHA will attenuate PAH-DNA adducts and lung carcinogenesis and tumorigenesis mediated by the PAHs BP and MC. Adult wild-type (WT) (A/J) mice, Cyp1a1-null, Cyp1a2-null, or Cyp1b1-null mice were exposed to PAHs benzo[a]pyrene (BP) or 3-methylcholanthrene (MC), and the effects of omega-3 fatty acid on PAH-mediated lung carcinogenesis and tumorigenesis were studied. The major findings were as follows: (i) omega-3 fatty acids significantly decreased PAH-DNA adducts in the lungs of each of the genotypes studied; (ii) decreases in PAH-DNA adduct levels by EPA/DHA was in part due to inhibition of CYP1B1; (iii) inhibition of soluble epoxide hydrolase (sEH) enhanced the EPA/DHA-mediated prevention of pulmonary carcinogenesis; and (iv) EPA/DHA attenuated PAH-mediated carcinogenesis in part by epigenetic mechanisms. Taken together, our results suggest that omega-3 fatty acids have the potential to be developed as cancer chemo-preventive agents in people.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Circadian control of cisplatin-DNA adduct repair and apoptosis in culture cells.

Cisplatin, a widely prescribed chemotherapeutic agent for treating solid tumors, induces DNA adducts and activates cellular defense mechanisms, including DNA repair, cell cycle checkpoint control, and apoptosis. Considering the circadian rhythmicity displayed by most chemotherapeutic agents and their varying therapeutic efficacy based on treatment timing, our study aimed to investigate whether the circadian clock system influences the DNA damage responses triggered by cisplatin in synchronized cells. We examined the DNA damage responses in circadian-synchronized wild-type mouse embryonic fibroblasts (WT-MEF; clock-proficient cells), cryptochrome1 and 2 double knock-out MEF (CRYDKO; clock-deficient cells), and mouse hepatocarcinoma Hepa1c1c7 cells. Varying the treatment time resulted in a significant difference in the rate of platinum-DNA adduct removal specifically in circadian-synchronized WT-MEF, while CRYDKO did not exhibit such variation. Moreover, diurnal variation in other DNA damage responses, such as cell cycle checkpoint activity indicated by p53 phosphorylation status and apoptosis measured by DNA break frequency, was observed only in circadian-synchronized WT-MEF, not in CRYDKO or mouse hepatocarcinoma Hepa1c1c7 cells. These findings highlight that the DNA damage responses triggered by cisplatin are indeed governed by circadian control exclusively in clock-proficient cells. This outcome bears potential implications for enhancing or devising chronotherapy approaches for cancer patients.

Effects of Diet on Aristolochic Acid-DNA Adduct Formation: Implications for Balkan Endemic Nephropathy Etiology.

Prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicine or AA-contaminated food is associated with the development of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), both public health risks to which the World Health Organization is calling for global action to remove exposure sources. The AA exposure-induced DNA damage is believed to be related to both the nephrotoxicity and carcinogenicity of AA observed in patients suffering from BEN. While the chemical toxicology of AA is well-studied, we investigated in this study the understated effect of different nutrients, food additives, or health supplements on DNA adduct formation by aristolochic acid I (AA-I). By culturing human embryonic kidney cells in an AAI-containing medium enriched with different nutrients, results showed that cells cultured in fatty acid-, acetic acid-, and amino acid-enriched media produced ALI-dA adducts at significantly higher frequencies than that cultured in the normal medium. ALI-dA adduct formation was most sensitive to amino acids, indicating that amino acid- or protein-rich diets might lead to a higher risk of mutation and even cancer. On the other hand, cells cultured in media supplemented with sodium bicarbonate, GSH, and NAC reduced ALI-dA adduct formation rates, which sheds light on their potential use as risk-mitigating strategies for people at risk of AA exposure. It is anticipated that the results of this study will help to better understand the effect of dietary habits on cancer and BEN development.

Formation of DHP-DNA Adducts from Rat Liver Microsomal Metabolism of 1,2-Unsaturated Pyrrolizidine Alkaloid-Containing Plant Extracts and Dietary Supplements.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are carcinogenic phytochemicals. We previously determined that carcinogenic PAs and PA N-oxides commonly form a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, namely, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4. This set of DHP-DNA adducts has been implicated as a potential biomarker of PA-induced liver tumor initiation from metabolism of individual carcinogenic PAs. To date, it is not known whether this generality occurs from metabolism of PA-containing plant extracts. In this study, we investigate the rat liver microsomal metabolism of nine PA-containing plant extracts and two PA-containing dietary supplements in the presence of calf thymus DNA. The presence of carcinogenic PAs and PA N-oxides in plant extracts was first confirmed by LC-MS/MS analysis with selected reaction monitoring mode. Upon rat liver microsomal metabolism of these PA-containing plant extracts and dietary supplements, the formation of this set of DHP-DNA adducts was confirmed. Thus, these results indicate that metabolism of PA-containing plant extracts and dietary supplements can generate DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, thereby potentially initiating liver tumor formation.

Aristolochic acid-containing Chinese herbal medicine and upper urinary tract urothelial carcinoma in Taiwan: a narrative review.

PURPOSE: The high incidence of upper urinary tract urothelial carcinoma (UTUC) in Taiwan is largely due to exposure to aristolochic acid (AA), a principal component of Aristolochia-based herbal medicines. Here we systematically review the molecular epidemiology, clinical presentation and biomarkers associated with AA-induced UTUC. METHODS: This is a narrative review. Medline, Embase, and Web of Science were searched from inception to December 31, 2021. Studies evaluating the association, detection, and clinical characteristics of AA and UTUC were included. RESULTS: A nationwide database revealed 39% of the Taiwanese population had been exposed to AA-containing herbs between 1997 and 2003. Epidemiological reports revealed AA posed a significantly higher hazard for renal failure and UTUC in herbalists and the general population who ingested AA-containing herbs. The presence of aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, located predominantly on the non-transcribed DNA strand, with a strong preference for deoxyadenosine in a consensus sequence (CAG), was observed in many UTUC patients. Clinically, AA-related UTUC patients were characterized by a younger age, female gender, impaired renal function and recurrence of contralateral UTUC. To date, there are no preventive measures, except prophylactic nephrectomy, for subjects at risk of AA nephropathy or AA-related UTUC. CONCLUSION: AA exposure via Aristolochia-based herbal medicines is a problem throughout Taiwan, resulting in a high incidence of UTUC. Aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, can be used as biomarkers to identify AA-related UTUC. AA-related UTUC is associated with a high recurrence rate of contralateral UTUC.

Black Raspberry Extract Enhances Glutathione Conjugation of the Fjord-Region Diol Epoxide Derived from the Tobacco Carcinogen Dibenzo[def,p]chrysene in Human Oral Cells.

In a series of previous studies we reported that black raspberry (BRB) powder inhibits dibenzo[a,l]pyrene (DBP)-induced DNA damage, mutagenesis, and oral squamous cell carcinoma (OSCC) development in mice. In the present study, using human oral leukoplakia (MSK-Leuk1) and squamous cell carcinoma (SCC1483) cells, we tested the hypothesis that BRB extract (BRBE) will enhance the synthesis of glutathione (GSH) and in turn increase GSH conjugation of the fjord-region DBP diol epoxide (DBPDE) derived from DBP leading to inhibition of DBP-induced DNA damage. The syntheses of DBPDE-GSH conjugate, DBPDE-dA adduct, and the corresponding isotope-labeled internal standards were performed; LC-MS/MS methods were used for their quantification. BRBE significantly (p < 0.05) increased cellular GSH by 31% and 13% at 6 and 24 h, respectively, in OSCC cells; in MSK-LeuK1 cells, the levels of GSH significantly (p < 0.05) increased by 55% and 22%, at 1 and 6 h. Since BRBE significantly enhanced the synthesis of GSH in both cell types, subsequent experiments were performed in MSK-Leuk1 cells. Western blot analysis was performed to determine the types of proteins involved in the synthesis of GSH. BRBE significantly (p < 0.05) increased the protein expression (2.5-fold) of the glutamate-cysteine ligase catalytic subunit (GCLC) but had no effect on the glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthetase (GSS). LC-MS/MS analysis showed that pretreatment of cells with BRBE followed by DBPDE significantly (p < 0.05) increased the levels of DBPDE-GSH conjugate (2.5-fold) and decreased DNA damage by 74% measured by assessing levels of DBPDE-dA adduct formation. Collectively, the results of this in vitro study clearly support our hypothesis, and the LC-MS/MS methods developed in the present study will be highly useful in testing the same hypothesis initially in our mouse model and ultimately in smokers.

Thermal-Responsive MXene-DNA Hydrogel for Near-Infrared Light Triggered Localized Photothermal-Chemo Synergistic Cancer Therapy.

Stimuli-responsive DNA hydrogels are promising candidates for cancer treatment, as they not only possess biocompatible and biodegradable 3D network structures as highly efficient carriers for therapeutic agents but also are capable of undergoing programmable gel-to-solution transition upon external stimuli to achieve controlled delivery. Herein, a promising platform for highly efficient photothermal-chemo synergistic cancer therapy is established by integrating DNA hydrogels with Ti3 C2 TX -based MXene as a photothermal agent and doxorubicin (DOX) as a loaded chemotherapeutic agent. Upon the irradiation of near-infrared light (NIR), temperature rise caused by photothermal MXene nanosheets triggers the reversible gel-to-solution transition of the DOX-loaded MXene-DNA hydrogel, during which the DNA duplex crosslinking structures unwind to release therapeutic agents for efficient localized cancer therapy. Removal of the NIR irradiation results in the re-formation of DNA duplex structures and the hydrogel matrix, and the recombination of free DOX and adaptive hydrogel transformations can also be achieved. As demonstrated by both in vitro and in vivo models, the MXene-DNA hydrogel system, with excellent biocompatibility and injectability, dynamically NIR-triggered drug delivery, and enhanced drug uptake under mild hyperthermia conditions, exhibits efficient localized cancer treatment with fewer side effects to the organisms.

Effects of Benzo[a]pyrene-DNA adducts, dietary vitamins, folate, and carotene intakes on preterm birth: a nested case-control study from the birth cohort in China.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) and its DNA adducts has been suggested to increase the risk of preterm birth (PB). Yet, few studies have been conducted to investigate this association, and the role of dietary nutrients intakes including vitamins, folate, and carotene during pre- and post-conception on this association has not been studied. METHODS: Building upon a birth cohort in Taiyuan China, we conducted a nested case control study including 83 PB and 82 term births. Benzo[a]pyrene (BaP)-DNA adducts were measured by an improved LC-MC/MC analytic method. Dietary nutrient intakes were estimated from food frequency questionnaire using the Chinese Standard Tables of Food Consumption. Multivariable logistic regression model was used to examine the associations. RESULTS: Increased risk of PB was observed as per interquartile increase in maternal BaP-DNA adduct level (OR = 1.27, 95%CI 0.95-1.67). Compared to low level (below mean) of maternal adducts, high level (above mean) of adducts was associated with the risk of PB (OR = 2.05, 95%CI 1.05-4.01). After stratified by dietary nutrients intakes, high adducts levels were associated with approximately 2-fourfold times increases in risk of PB among women with low vitamin A, C, E, folate, and carotene intakes during pre- and/or post-conception. Stronger stratified associations were consistently seen during preconception. Similar patterns were observed after additional adjustment for supplementation. CONCLUSIONS: Our study supports the hypothesis that high level of maternal PAHs exposure was significantly associated with increased risk of PB, and provides the first evidence that dietary vitamins, carotene, and folate intake levels may modify this association during different pregnancy windows. Our findings are relevant to identify recommendation for environment management and prenatal nutrition regarding pregnant women and newborns. Further investigation in other populations is warranted.

Comprehensive profile of DNA adducts as both tissue and urinary biomarkers of exposure to acrylamide and chemo-preventive effect of catechins in rats.

Two representative DNA adducts from acrylamide exposure, N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl) adenine (N3-GA-Ade), are important long-term exposure biomarkers for evaluating genotoxicity of acrylamide. Catechins as natural antioxidants present in tea possess multiple health benefits, and may also have the potential to protect against acrylamide-induced DNA damage. The current study developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous analysis of N7-GA-Gua and N3-GA-Ade in tissues and urine. The validated UHPLC-MS/MS method showed high sensitivity, with limit of detection and limit of quantification ranging 0.2-0.8 and 0.5-1.5 ng/mL, respectively, and achieved qualified precision (RSD<14.0%) and spiking recovery (87.2%-110.0%) with elution within 6 min, which was suitable for the analysis of the two DNA adducts in different matrices. The levels of N7-GA-Gua and N3-GA-Ade ranged 0.9-11.9 and 0.6-3.5 µg/g creatinine in human urine samples, respectively. To investigate the interventional effects of catechins on the two DNA adducts from acrylamide exposure, rats were supplemented with three types of catechins (tea polyphenols, epigallocatechin gallate, and epicatechin) 30 min before administration with acrylamide. Our results showed that catechins effectively inhibited the formation of DNA adducts from acrylamide exposure in both urine and tissues of rats. Among three catechins, epicatechin performed the best inhibitory effect. The current study provided evidence for the chemo-preventive effect of catechins, indicating that dietary supplement of catechins may contribute to health protection against exposure to acrylamide.

Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy.

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation.

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells.

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3'-yl)-2'-desoxyguanosine (E3'N2dG) and N6-(isoestragole-3'-yl)-desoxyadenosine (E3'N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3'N6dA at 10 µM, E3'N2dG at 1µM estragole). E3'N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 µM after 24 h and < 0.5 µM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 µM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 µM estragole led to a 3.2 fold and 300 µM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a 'practical threshold' dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.

[Quantification of aristolochic acids and their DNA adducts in mice kidney and liver by HPLC-MS/MS].

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 µm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.

Aristolochic acid and its effect on different cancers in uro-oncology.

PURPOSE OF REVIEW: To acquaint urologists with aristolochic acid nephropathy, an iatrogenic disease that poses a distinct threat to global public health. In China alone, 100 million people may currently be at risk. We illustrate the power of molecular epidemiology in establishing the cause of this disease. RECENT FINDINGS: Molecular epidemiologic approaches and novel mechanistic information established a causative linkage between exposure to aristolochic acid and urothelial carcinomas of the bladder and upper urinary tract. Noninvasive tests are available that detect urothelial cancers through the genetic analysis of urinary DNA. Combined with cytology, some of these tests can detect 95% of patients at risk of developing bladder and/or upper urothelial tract cancer. Robust biomarkers, including DNA-adduct and mutational signature analysis, unequivocally identify aristolochic acid-induced tumours. The high mutational load associated with aristolochic acid-induced tumours renders them candidates for immune-checkpoint therapy. SUMMARY: Guided by recent developments that facilitate early detection of urothelial cancers, the morbidity and mortality associated with aristolochic acid-induced bladder and upper tract urothelial carcinomas may be substantially reduced. The molecular epidemiology tools that define aristolochic acid-induced tumours may be applicable to other studies assessing potential environmental carcinogens.

Genomics of aging: Genes, adducts, and telomeres.

Genomics influences the aging process in many different ways. This 10-part series of articles describes what is known about genetics and aging, including genes, adducts, and telomeres, decreased immune defenses, oxidation and inefficient mitochondria, toxins and radiation, glycosylation, caloric intake and sirtuin production, neurotransmitter imbalance, hormone mechanisms, reduced nitric oxide, and stem cell slowdown. This first article explores gene adducts as an epigenetic "sludge," the influence of telomeres and other mutations that contribute to DNA dysfunction, cell stress, and premature aging. Factors that contribute to adduct formation and reduced telomere length are presented along with some changes in behavior, environmental exposure, food/supplement use, weight, sleep, and exercise that have been found to reduce damage, potentially increasing longevity. Adherence to a Mediterranean diet that contains fruits and whole grains along with fiber, antioxidants (e.g., beta-carotene, vitamins C and E), omega-3 fatty acids, and soy protein may reduce DNA adducts and protect telomeres. So providers may want to recommend these simple but key clinical and individual changes to enhance DNA health, wellness, and longevity.

Oral Dosing of Dihydromethysticin Ahead of Tobacco Carcinogen NNK Effectively Prevents Lung Tumorigenesis in A/J Mice.

Our early studies demonstrated an impressive chemopreventive efficacy of dihydromethysticin (DHM), unique in kava, against tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice in which DHM was supplemented in the diet. The current work was carried out to validate the efficacy, optimize the dosing schedule, and further elucidate the mechanisms using oral bolus dosing of DHM. The results demonstrated a dose-dependent chemopreventive efficacy of DHM (orally administered 1 h before each of the two NNK intraperitoneal injections, 1 week apart) against NNK-induced lung adenoma formation. Temporally, DHM at 0.8 mg per dose (∼32 mg per kg body weight) exhibited 100% lung adenoma inhibition when given 3 and 8 h before each NNK injection and attained >93% inhibition when dosed at either 1 or 16 h before each NNK injection. The simultaneous treatment (0 h) or 40 h pretreatment (-40 h) decreased lung adenoma burden by 49.8% and 52.1%, respectively. However, post-NNK administration of DHM (1-8 h after each NNK injection) was ineffective against lung tumor formation. In short-term experiments for mechanistic exploration, DHM treatment reduced the formation of NNK-induced O6-methylguanine (O6-mG, a carcinogenic DNA adduct in A/J mice) in the target lung tissue and increased the urinary excretion of NNK detoxification metabolites as judged by the ratio of urinary NNAL-O-gluc to free NNAL, generally in synchrony with the tumor prevention efficacy outcomes in the dose scheduling time-course experiment. Overall, these results suggest DHM as a potential chemopreventive agent against lung tumorigenesis in smokers, with O6-mG and NNAL detoxification as possible surrogate biomarkers.

Facile Construction of i-Motif DNA-Conjugated Gold Nanostars as Near-Infrared and pH Dual-Responsive Targeted Drug Delivery Systems for Combined Cancer Therapy.

Stimuli-responsive DNA-based nanostructures have emerged as promising vehicles for intelligent drug delivery. In this study, i-motif DNA-conjugated gold nanostars (GNSs) were fabricated in a facile manner as stimuli-responsive drug delivery systems (denoted as A-GNS/DNA/DOX) for the treatment of cancer via combined chemo-photothermal therapy. The i-motif DNA is sensitive to the environmental pH and can switch from a single-stranded structure to a C-tetrad (i-motif) structure as the environmental pH decreases from neutral (∼7.4) to acidic (<6.0). The loaded drug can then be released along with the conformational changes. To enhance cellular uptake and improve cancer cell selectivity, the aptamer AS1411, which recognizes nucleolins, was employed as a targeting moiety. The A-GNS/DNA/DOX nanocomposites were found to be highly capable of photothermal conversion and exhibited photostability under near-infrared (NIR) irradiation, and the pH and NIR irradiation effectively triggered the drug-release behaviors. In addition, the A-GNS/DNA/DOX nanocomposites exhibited good biocompatibility. The targeting recognition enabled the A-GNS/DNA/DOX to exhibit higher cellular uptake and therapeutic efficiency than the GNS/DNA/DOX. Notably, under NIR irradiation, a synergistic effect between chemotherapy and photothermal therapy can be achieved with the proposed delivery system, which exhibits much higher therapeutic efficiency both in monolayer cancer cells and tumor spheroids as compared with a single therapeutic method. This study highlights the potential of GNS/DNA nanoassemblies for intelligent anticancer drug delivery and combined cancer therapy.

Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

Loss of NQO1 generates genotoxic estrogen-DNA adducts in Fuchs Endothelial Corneal Dystrophy.

Fuchs Endothelial Corneal Dystrophy (FECD) is an age-related genetically complex disease characterized by increased oxidative DNA damage and progressive degeneration of corneal endothelial cells (HCEnCs). FECD has a greater incidence and advanced phenotype in women, suggesting a possible role of hormones in the sex-driven differences seen in the disease pathogenesis. In this study, catechol estrogen (4-OHE2), the byproduct of estrogen metabolism, induced genotoxic estrogen-DNA adducts formation, macromolecular DNA damage, and apoptotic cell death in HCEnCs; these findings were potentiated by menadione (MN)-mediated reactive oxygen species (ROS). Expression of NQO1, a key enzyme that neutralizes reactive estrogen metabolites, was downregulated in FECD, indicating HCEnC susceptibility to reactive estrogen metabolism in FECD. NQO1 deficiency in vitro exacerbated the estrogen-DNA adduct formation and loss of cell viability, which was rescued by the supplementation of N-acetylcysteine, a ROS scavenger. Notably, overexpression of NQO1 in HCEnCs treated with MN and 4-OHE2 quenched the ROS formation, thereby reducing the DNA damage and endothelial cell loss. This study signifies a pivotal role for NQO1 in mitigating the macromolecular oxidative DNA damage arising from the interplay between intracellular ROS and impaired endogenous estrogen metabolism in post-mitotic ocular tissue cells. A dysfunctional Nrf2-NQO1 axis in FECD renders HCEnCs susceptible to catechol estrogens and estrogen-DNA adducts formation. This novel study highlights the potential role of NQO1-mediated estrogen metabolite genotoxicity in explaining the higher incidence of FECD in females.