DNA Adduct Formation Research Articles

The Intercalator Ethidium Bromide Generates Covalent Adducts at Apurinic/Apyrimidinic Sites in DNA.

Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA. Adduct formation proceeds via the reaction of the amino group(s) on ethidium with the ring-opened aldehyde residue of the AP site in DNA to yield an imine. Ethidium-AP adducts may form under a variety of circumstances due to the ubiquitous occurrence of AP sites in cellular and synthetic DNA.

Apigenin attenuates cisplatin-induced hair cell damage in the zebrafish lateral line

Cisplatin, a widely used chemotherapy drug, is notorious for causing ototoxicity, which leads to irreversible sensorineural hearing loss by damaging cochlear sensory hair cells (HCs), spiral ganglion neurons (SGNs), and the stria vascularis (SV). Mechanisms include DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammation, ultimately triggering cell death pathways like apoptosis, necroptosis, pyroptosis, or ferroptosis. Apigenin, a natural flavonoid found in various foods and beverages, possesses antioxidant, anti-inflammatory, and anti-tumor properties. Despite these benefits, its potential to mitigate cisplatin-induced ototoxicity remains unexplored. To investigate, we administered varying concentrations of apigenin (1 μM, 20 μM, 100 μM, and 250 μM) alongside cisplatin (200 μM) to zebrafish larvae at 5 days post fertilization. Cisplatin significantly reduced lateral line HCs, impacting auditory function as shown in startle response tests. However, co-administration with apigenin preserved lateral line HCs and mitigated cisplatin-induced hearing loss. In larvae exposed to cisplatin, TUNEL assay confirmed significant HCs apoptosis, which apigenin effectively countered by suppressing reactive oxygen species accumulation in lateral line HCs. RNA-seq analysis highlighted apigenin's role in modulating apoptosis-related pathways, supporting its protective effects against cisplatin-induced ototoxicity. These findings underscore apigenin's potential as a crucial protective agent against cisplatin-induced ototoxicity, meriting further investigation for clinical applications.

Whole Genome Sequencing Analysis of Model Organisms Elucidates the Association Between Environmental Factors and Human Cancer Development

Determining a novel etiology and mechanism of human cancer requires extraction of characteristic mutational signatures derived from chemical substances. This study explored the mutational signatures of N-nitroso bile acid conjugates using Salmonella strains. Exposing S. typhimurium TA1535 to N-nitroso-glycine/taurine bile acid conjugates induced a predominance of C:G to T:A transitions. Two mutational signatures, B1 and B2, were extracted. Signature B1 is associated with N-nitroso-glycine bile acid conjugates, while Signature B2 is linked to N-nitroso-taurine bile acid conjugates. Signature B1 revealed a strong transcribed strand bias with GCC and GCT contexts, and the mutation pattern of N-nitroso-glycine bile acid conjugates in YG7108, which lacks O6-methylguanine DNA methyltransferases, matched that of the wild-type strain TA1535, suggesting that O6-methyl-deoxyguanosine contributes to mutations in the relevant regions. COSMIC database-based similarity analysis revealed that Signature B1 closely resembled SBS42, which is associated with occupational cholangiocarcinoma caused by overexposure to 1,2-dichlolopropane (1,2-DCP) and/or dichloromethane (DCM). Moreover, the inflammatory response pathway was induced by 1,2-DCP exposure in a human cholangiocyte cell line, and iNOS expression was positive in occupational cholangiocarcinomas. These results suggest that 1,2-DCP triggers an inflammatory response in biliary epithelial cells by upregulating iNOS and N-nitroso-glycine bile acid conjugate production, resulting in cholangiocarcinoma via DNA adduct formation.

Use of physiologically based kinetic modeling to predict neurotoxicity and genotoxicity of methylglyoxal in humans

This study aimed to evaluate human neurotoxicity and genotoxicity risks from dietary and endogenous methylglyoxal (MGO), utilizing physiologically based kinetic (PBK) modeling-facilitated reverse dosimetry as a new approach methodology (NAM) to extrapolate in vitro toxicity data to in vivo dose-response predictions. A human PBK model was defined based on a newly developed and evaluated mouse model enabling the translation of in vitro toxicity data for MGO from human stem cell-derived neurons and WM-266-4 melanoma cells into quantitative human in vivo toxicity data and subsequent risk assessment by the margin of exposure (MOE) approach. The results show that the MOEs resulting from daily dietary intake did not raise a concern for endpoints for neurotoxicity including mitochondrial function, cytotoxicity, and apoptosis, while those for DNA adduct formation could not exclude a concern over genotoxicity. Endogenous MGO formation, especially under diabetic conditions, resulted in MOEs that raised concern not only for genotoxicity but also for some of the neurotoxicity endpoints evaluated. Thus, the results also point to the importance of taking the endogenous levels into account in the risk assessment of MGO.

Evidence for the formation of 6PPD-quinone from antioxidant 6PPD by cytochrome P450

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) as a rubber antioxidant has attracted global concern, since its ozone-oxidation product 6PPD-quinone (6PPDQ) was found to be the primary toxicant responsible for urban runoff mortality syndrome in coho salmon. However, the biotransformation fate and associated toxicological mechanism of 6PPD have not received much study yet. In this work, the in vitro assays showed 6PPD can be transformed into 6PPDQ by cytochromes P450 (CYP450) in human liver microsomes (HLMs) with 0.98 % production rate, and the adducts of 6PPDQ with calf thymus DNA and the N-N coupling product between 6PPD and 6PPDQ were further identified after 6PPD incubation in HLMs. Further evidence for the 6PPDQ formation can be obtained from the in vivo assays that the 6PPDQ-DNA adducts and 6PPD-N-N-6PPDQ dimer were detected in mice by oral gavage with 6PPD, and the latter dimer species was detected as well in 6PPD exposure to zebrafish larvae. Especially, the bioaccumulation property and high reactivity of 6PPDQ result in the continuous formation of the significant DNA adducts and 6PPD-N-N-6PPDQ dimer even in case of low production rate of biotransformation of 6PPD to 6PPDQ, which may provide potentially effective biomarkers for such process. DFT computations revealed the formation mechanism of 6PPDQ is the (N)H-abstraction of 6PPD by CYP450, followed by amino radical rebound at the nearby ortho-carbon, yielding a quinol intermediate due to spin delocalization, that might readily undergo further oxidation by CYP450 into 6PPDQ.

Ultra high-performance liquid chromatography tandem mass spectrometry: Development and validation of an analytical method for N2-Ethyl-2’-deoxyguanosine in dried blood spot

In the body, ethanol is metabolized to acetaldehyde by alcohol dehydrogenase and CYP2E1. Acetaldehyde is the main carcinogen that forms DNA adducts, N2-Ethylidene-2'-deoxyguanosine (N2-Ethylidene-dG), which can cause DNA damage and lead to cancer. However, N2-Ethylidene-dG is an unstable form at the nucleoside level and is difficult to measure. So it needs to be reduced using NaBH4 to become N2-Ethyl-2'-deoxyguanosine (N2-Et-dG). This study aims to obtain a selective, sensitive, and validated analytical method to determine N2-Et-dG using ultra-high-performance liquid chromatography-tandem mass spectrometry with allopurinol as the internal standard. The N2-Et-dG analysis was performed using a C18 Acquity® Bridged Ethylene Hybrid column of (1.7 μm, 100 mm × 2.1 mm). The sample matrix used was dried blood spots, and then the DNA sample was extracted using the QIAamp DNA Mini Kit. The optimum analysis conditions were obtained in a combination eluent of 0.1 % acetic acid and methanol (60:40 v/v) with a flow rate of 0.1 mL/min and eluted isocratically for 5 min. Quantification analysis was performed using triple quadrupole mass spectrometry with electrospray ionization in positive mode, with detection at m/z 296.16 > 180.16 for N2-Et-dG and 137.03 > 110.05 for allopurinol, respectively. The validated analytical method follows the 2018 USA Food and Drug Administration guidelines with a linear concentration range of 5–200 ng/mL.

Aerosolized e-liquid base constituents induce cytotoxicity and genotoxicity in oral keratinocytes.

Electronic cigarette (e-cigarette) use among adults in the United States continues to rise. Particularly concerning is the impact of e-cigarette aerosol inhalation on the oral mucosa. Aerosols are derived from a heated e-liquid base of propylene glycol/glycerin (PG/G) often mixed with nicotine and chemical flavors. Of note, harmful and potentially harmful constituents (HPHCs), including metals and volatile organic compounds, have been detected in e-cigarette aerosols. It remains unknown, however, whether aerosols exclusively derived from e-liquid PG/G are detrimental to oral keratinocytes. The present study analyzed toxicological outcomes in normal oral keratinocytes exposed to model nicotine-free, unflavored PG/G e-liquid aerosols. Cell viability/cytotoxicity, genotoxicity, and immunoblotting assays were conducted in NOKSI, a gingiva-derived oral keratinocyte cell line, following exposure to model e-liquid aerosols or non-aerosolized controls. The HPHC acrolein, reported to form DNA adducts in the buccal mucosa from e-cigarette users, was also used in similar assays. PG/G e-liquid aerosol extracts significantly enhanced cytotoxic and DNA damaging responses in NOKSI cells when compared to non-aerosolized e-liquid treatment. Acrolein treatment led to similar results. The aerosolization process of PG/G e-liquid is a critical determinant of marked cytotoxic and genotoxic stimuli in oral keratinocytes.

Positive Feedback Mechanism in Aristolochic Acid I Exposure-Induced Anemia and DNA Adduct Formation: Implications for Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a chronic kidney disease that predominantly affects inhabitants of rural farming communities along the Danube River tributaries in the Balkans. Long-standing research has identified dietary exposure to aristolochic acids (AAs) as the principal toxicological cause. This study investigates the pathophysiological role of anemia in BEN, noting its earlier and more severe manifestation in BEN patients compared to those with other chronic kidney diseases. Utilizing a mouse model, our research demonstrates that prolonged exposure to aristolochic acid I (AA-I) (the most prevalent AA variant) leads to significant red blood cell depletion through DNA damage, such as DNA adduct formation in bone marrow, prior to observable kidney function decline. Furthermore, in vitro experiments with kidney cells exposed to lowered oxygen and pH conditions mimicking an anemia environment show enhanced DNA adduct formation, suggesting increased AA-I mutagenicity and carcinogenicity. These findings indicate for the first time a positive feedback mechanism of AA-induced anemia, DNA damage, and kidney impairment in BEN progression. These results not only advance our understanding of the underlying mechanisms of BEN but also highlight anemia as a potential target for early BEN diagnosis and therapy.

Enzymatic Acrolein Production System and Its Impact on Human Cells.

Acrolein is an environmental toxicant and is also generated by microbial metabolism in the intestinal tract. Aqueous acrolein rapidly dissipates from standard human cell culture media with nondetectable levels after 8 h, hindering cell-based studies to understand its biological impacts. Thus, we developed an extracellular acrolein biosynthesis system to continuously produce acrolein compatible with human cell culture conditions. The approach uses spermine as a precursor, amine oxidase found in fetal calf serum, and catalase to remove the hydrogen peroxide byproduct. We confirmed amine oxidase activity of calf serum using a colorimetric assay and further tested the requirement for catalase in the system to mitigate hydrogen peroxide-induced cytotoxicity. We calibrated responses of human colon cells to this enzymatic acrolein production system by comparing transcriptional responses, DNA adduct formation and cytotoxicity responses to either this system or pure acrolein exposures in a human colon cell line. Several genes related to oxidative stress including HMOX1, and the colorectal cancer-related gene SEMA4A were upregulated similarly between the enzymatic acrolein production system or pure acrolein. The acrolein-DNA adduct γ-OH-Acr-dG increased in a dose-dependent manner with spermine in the enzymatic acrolein production system, producing a maximum of 1065 adducts per 108 nucleosides when 400 μM spermine was used. This biosynthetic production method provides a relevant model for controlled acrolein exposure in cultured human cells and overcomes current limitations due to its physical properties and limited availability.

Novel insights into the role of bisphenol A (BPA) in genomic instability.

Bisphenol A (BPA) is a phenolic chemical that has been used for over 50 years in the manufacturing of polycarbonate and polyvinyl chloride plastics, and it is one of the highest volume chemicals produced worldwide. Because BPA can bind to and activate estrogen receptors, studies have mainly focused on the effect of BPA in disrupting the human endocrine and reproductive systems. However, BPA also plays a role in promoting genomic instability and has been associated with initiating carcinogenesis. For example, it has been recently shown that exposure to BPA promotes the formation of single stranded DNA gaps, which may be associated with increased genomic instability. In this review, we outline the mechanisms by which BPA works to promote genomic instability including chromosomal instability, DNA adduct formation, ROS production, and estrogen receptor (ER) activation. Moreover, we define the ways in which BPA promotes both carcinogenesis and resistance to chemotherapy, and we provide critical insights into future directions and outstanding questions in the field.

Combustion-Derived Pollutants Linked with Kidney Disease in Low-Lying Flood-Affected Areas in the Balkans.

Tens of thousands of people in southern Europe suffer from Balkan endemic nephropathy (BEN), and four times as many are at risk. Incidental ingestion of aristolochic acids (AAs), stemming from the ubiquitousAristolochia clematitis(birthwort) weed in the region, leads to DNA adduct-induced toxicity in kidney cells, the primary cause of BEN. Numerous cofactors, including toxic organics and metals, have been investigated, but all have shown small contributions to the overall BEN relative to non-BEN village distribution gradients. Here, we reveal that combustion-derived pollutants from wood and coal burning in Serbia also contaminate arable soil and test as plausible causative factors of BEN. Using a GC-MS screening method, biomass-burning-derived furfural and coal-burning-derived medium-chain alkanes were detected in soil samples from BEN endemic areas levels at up to 63-times and 14-times higher, respectively, than in nonendemic areas. Significantly higher amounts were also detected in colocated wheat grains. Coexposure studies with cultured kidney cells showed that these pollutants enhance DNA adduct formation by AA, - the cause of AA nephrotoxicity and carcinogenicity. With the coincidence of birthwort-derived AAs and the widespread practice of biomass and coal burning for household cooking and heating purposes and agricultural burning in rural low-lying flood-affected areas in the Balkans, these results implicate combustion-derived pollutants in promoting the development of BEN.

From single DNA adducts measurement to DNA adductomics in molecular epidemiology of cancer.

Environmental carcinogens exert their carcinogenic effects by forming DNA adducts. This type of DNA damage can also be formed endogenously as a result of, e.g., oxidative damage. Unrepaired DNA adducts may induce mutations in critical genes, leading to the initiation of chemical carcinogenesis. Therefore, detection, identification, and quantification of DNA adducts is essential for cancer risk assessment. Over the last 50 years, the major DNA adducts formed by different classes of environmental carcinogens were characterized. With the development of techniques such as 32P-postlabeling, their measurement was implemented into molecular epidemiology. Advances in liquid chromatography-tandem mass spectrometry (LC-MS ) made the measurement of adducts more precise and allowed to gain knowledge about their identity and structures. Therefore, opened the way to DNA adductomics, the "omics" approach investigating DNA adducts comprehensively, similarly to proteomics. This review presents the historical perspective of DNA adducts research and the emerging field of adductomics.

The detoxification ability of sex-role reversed seahorses determines the sexual dimorphism in immune responses to benzo[a]pyrene exposure

Sexual dimorphism in immune responses is an essential factor in environmental adaptation. However, the mechanisms involved remain obscure owing to the scarcity of data from sex-role-reversed species in stressed conditions. Benzo[a]pyrene (BaP) is one of the most pervasive and carcinogenic organic pollutants in coastal environments. In this study, we evaluated the potential effects on renal immunotoxicity of the sex-role-reversed lined seahorse (Hippocampus erectus) toward environmental concentrations BaP exposure. Our results discovered the presence of different energy-immunity trade-off strategies adopted by female and male seahorses during BaP exposure. BaP induced more severe renal damage in female seahorses in a concentration-dependent manner. BaP biotransformation and detoxification in seahorses resemble those in mammals. Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and 9-hydroxybenzo[a]pyrene (9-OH-BaP) formed DNA adducts and disrupted Ca2+ homeostasis may together attribute the renal immunotoxicity. Sexual dimorphisms in detoxification of both BPDE and 9-OH-BaP, and in regulation of Ca2+, autophagy and inflammation, mainly determined the extent of renal damage. Moreover, the mechanism of sex hormones regulated sexual dimorphism in immune responses needs to be further elucidated. Collectively, these findings contribute to the understanding of sexual dimorphism in the immunotoxicity induced by BaP exposure in seahorses, which may attribute to the dramatic decline in the biodiversity of the genus.

DNA adduct formation in Saccharomyces cerevisiae following exposure to environmental pollutants, as in vivo model for molecular toxicity studies.

DNA adduction in the model yeast Saccharomyces cerevisiae was investigated after exposure to the fungicide penconazole and the reference genotoxic compound benzo(a)pyrene, for validating yeasts as a tool for molecular toxicity studies, particularly of environmental pollution. The effect of the toxicants on the yeast's growth kinetics was determined as an indicator of cytotoxicity. Fermentative cultures of S. cerevisiae were exposed to 2ppm of Penconazole during different phases of growth; while 0.2 and 2ppm of benzo(a)pyrene were applied to the culture medium before inoculation and on exponential cultures. Exponential respiratory cultures were also exposed to 0.2ppm of B(a)P for comparison of both metabolisms. Penconazole induced DNA adducts formation in the exponential phase test; DNA adducts showed a peak of 54.93 adducts/109 nucleotides. Benzo(a)pyrene induced the formation of DNA adducts in all the tests carried out; the highest amount of 46.7 adducts/109 nucleotides was obtained in the fermentative cultures after the exponential phase exposure to 0.2ppm; whereas in the respiratory cultures, 14.6 adducts/109 nucleotides were detected. No cytotoxicity was obtained in any experiment. Our study showed that yeast could be used to analyse DNA adducts as biomarkers of exposure to environmental toxicants.

Environmental profiles, hazard identification, and toxicological hallmarks of emerging tire rubber-related contaminants 6PPD and 6PPD-quinone

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is commonly used in rubber compounds as antioxidants to protect against degradation from heat, oxygen, and ozone exposure. This practice extends the lifespan of rubber products, including tires, by preventing cracking, aging, and deterioration. However, the environmental consequences of waste generated during rubber product use, particularly the formation of 6PPD-quinone (6PPD-Q) through the reaction of 6PPD with ozone, have raised significant concerns due to their detrimental effects on ecosystems. Extensive research has revealed the widespread occurrence of 6PPD and its derivate 6PPD-Q in various environmental compartments, including air, water, and soil. The emerging substance of 6PPD-Q has been shown to pose acute mortality and long-term hazards to aquatic and terrestrial organisms at concentrations below environmentally relevant levels. Studies have demonstrated toxic effects of 6PPD-Q on a range of organisms, including zebrafish, nematodes, and mammals. These effects include neurobehavioral changes, reproductive dysfunction, and digestive damage through various exposure pathways. Mechanistic insights suggest that mitochondrial stress, DNA adduct formation, and disruption of lipid metabolism contribute to the toxicity induced by 6PPD-Q. Recent findings of 6PPD-Q in human samples, such as blood, urine, and cerebrospinal fluid, underscore the importance of further research on the public health and toxicological implications of these compounds. The distribution, fate, biological effects, and underlying mechanisms of 6PPD-Q in the environment highlight the urgent need for additional research to understand and address the environmental and health impacts of these compounds.

Amino acid‐decorated mesoporous silica nanoparticles loaded with titanocene derivatives for targeted anticancer studies

Nanostructured materials possess promising potential for cancer therapy through precise adjustment of their functionalization and physicochemical attributes. This study primarily focuses on the synthesis and characterization of mesoporous silica nanoparticles (MSN) that are functionalized with titanocene dichloride (a therapeutic agent) and one of several amino acids—cysteine, captopril, penicillamine, or methionine—utilizing 3‐aminopropyltriethoxysilane (AP) as a linker. This synthesis yielded four innovative metallodrug‐functionalized nanostructured materials (MSN‐AP‐Cys‐Ti, MSN‐AP‐Cap‐Ti, MSN‐AP‐Pen‐Ti, and MSN‐AP‐Met‐Ti), meticulously characterized using diverse analytical techniques such as X‐ray diffraction (XRD), X‐ray fluorescence (XRF), diffuse reflectance ultraviolet–visible (DR UV–Vis), Fourier transform infrared (FTIR), solid‐state nuclear magnetic resonance (NMR) spectroscopy, and transmission electron microscopy (TEM). The textural properties of the nanomaterials post‐functionalization displayed slight modifications, confirming the successful integration of the therapeutic agents. Evaluation of cytotoxicity in the breast cancer cell line MDA‐MB‐231, with the healthy cell line Hek‐293T as control via MTT assays, revealed the active nature of the functionalized silica‐based materials. The viability of both cell lines indicated a concentration‐dependent response to the materials. Among the tested systems, cysteine and captopril exhibited the highest activity concerning IC50 relative to material concentration. The enhanced biological activity of higher functionalized nanosystems suggests a favorable cell internalization facilitated by the amino acid fragment. Additionally, qualitative DNA binding studies hinted at potential DNA adsorption on the surface of the metallodrug‐functionalized nanomaterials, forming DNA adducts where a strand of DNA covalently bonds to the metallodrug moiety. This was deduced from the hypsochromic shift in absorbance of the characteristic π–π* and n–π* transitions in DNA, which occurred from 1.01 to 0.76 and 1.26–0.19 following drug (MSN‐AP‐Cap‐Ti) interaction.

Formation of DNA Adducts by 1-Methoxy-3-indolylmethylalcohol, a Breakdown Product of a Glucosinolate, in the Mouse: Impact of the SULT1A1 Status-Wild-Type, Knockout or Humanised.

We previously found that feeding rats with broccoli or cauliflower leads to the formation of characteristic DNA adducts in the liver, intestine and various other tissues. We identified the critical substances in the plants as 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate and its degradation product 1-MIM-OH. DNA adduct formation and the mutagenicity of 1-MIM-OH in cell models were drastically enhanced when human sulfotransferase (SULT) 1A1 was expressed. The aim of this study was to clarify the role of SULT1A1 in DNA adduct formation by 1-MIM-OH in mouse tissues in vivo. Furthermore, we compared the endogenous mouse Sult1a1 and transgenic human SULT1A1 in the activation of 1-MIM-OH using genetically modified mouse strains. We orally treated male wild-type (wt) and Sult1a1-knockout (ko) mice, as well as corresponding lines carrying the human SULT1A1-SULT1A2 gene cluster (tg and ko-tg), with 1-MIM-OH. N2-(1-MIM)-dG and N6-(1-MIM)-dA adducts in DNA were analysed using isotope-dilution UPLC-MS/MS. In the liver, caecum and colon adducts were abundant in mice expressing mouse and/or human SULT1A1, but were drastically reduced in ko mice (1.2-10.6% of wt). In the kidney and small intestine, adduct levels were high in mice carrying human SULT1A1-SULT1A2 genes, but low in wt and ko mice (1.8-6.3% of tg-ko). In bone marrow, adduct levels were very low, independently of the SULT1A1 status. In the stomach, they were high in all four lines. Thus, adduct formation was primarily controlled by SULT1A1 in five out of seven tissues studied, with a strong impact of differences in the tissue distribution of mouse and human SULT1A1. The behaviour of 1-MIM-OH in these models (levels and tissue distribution of DNA adducts; impact of SULTs) was similar to that of methyleugenol, classified as "probably carcinogenic to humans". Thus, there is a need to test 1-MIM-OH for carcinogenicity in animal models and to study its adduct formation in humans consuming brassicaceous foodstuff.

Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

Abstract 1880: Ecubectedin and PM54 demonstrate antitumor activity in patient-derived xenograft models of soft tissue sarcoma

Abstract Objective: Soft tissue sarcoma (STS) is a heterogenous group of rare, malignant tumors arising from mesenchymal tissue. Doxorubicin (DOX)-based chemotherapy is the standard for advanced or metastatic STS, despite low response rates and poor disease control. We explored the antitumor activity of two novel compounds, ecubectedin (ECU; PM14) and PM54 (PharmaMar), in two patient-derived xenograft (PDX) models of STS, leiomyosarcoma (LMS) and CIC-rearranged sarcoma (CRS). Mode of action of ECU and PM54 is similar to lurbinectedin (LUR), inhibiting oncogenic transcription by eviction of oncogenic transcription factors from gene promotors’ recognition sequences, while inducing the generation of double strand breaks through DNA adduct formation. Methods: Female NMRI nu/nu mice (n=104) were transplanted bilaterally with STS PDX: UZLX-STS134 (CRS) and UZLX-STS22_2 (LMS). Xenografted mice were randomized to six treatment groups: 1) vehicle (VEH) (5% dextrose) 5 mL/kg once weekly (QW) intravenously (IV); 2) DOX 5 mg/kg QW IV; 3) trabectedin (TRA) 0.15 mg/kg QW IV; 4) LUR 0.18 mg/kg QW IV; 5) ECU 1.2 mg/kg QW IV; 6) PM54 1.2 mg/kg QW IV. Treatment lasted 16 days and antitumor activity was assessed by tumor volume (TV) analysis, histopathology and western blot. The Mann-Whitney U test was used to compare groups, the Wilcoxon test to compare to baseline. Results: ECU and PM54 caused TV shrinkage in UZLX-STS134, while TV in other groups grew significantly on day 16 (Table 1). VEH treatment led to the largest TV increase on day 16, compared to any treatment. PM54 treatment showed TV stabilization in UZLX-STS22_2, and significantly smaller tumors than VEH and TRA treated tumors (p<0.05 and p<0.01, respectively). Histopathological evaluation corroborate the results from the relative TV analysis. Conclusion: Novel drugs exhibit promising results in the PDX models tested, supporting ongoing exploration in additional PDX models of synovial sarcoma and dedifferentiated liposarcoma. Table 1: TV change on day 16 versus baseline, and comparison of proliferative and apoptotic activity Tumor volume change Proliferative activity (pHH3) Apoptotic activity (cPARP) Subtypes UZLX-STS134 UZLX-STS22_2 UZLX-STS134 UZLX-STS22_2 UZLX-STS134 UZLX-STS22_2 VEH(5 ML/kg) 387%*** 349%** n/a n/a n/a n/a DOX (5 mg/kg/QW IV) 177%*** 243%** ↓*** ↓ ↑** ↑ TRA(0.15 mg/kg/QW IV) 230%** 254%*** ↓** ↓** ↑** ↑*** LUR(0.18 mg/kg/QW IV) 202%** 232%** ↓*** ↓*** ↑*** ↑** ECU(1.2 mg/kg/QW IV) 41%*** 181%** ↓*** ↓*** ↑*** ↑*** PM54 (1.2 mg/kg/QW IV) 76%*** 162% ↓*** ↓*** ↑*** ↑ Citation Format: Daniël Gorgels, Agnieszka Wozniak, Chao-Chi Wang, Pablo M. Avilés, Maria J. Guillen, Carmen Cuevas, Patrick Schöffski. Ecubectedin and PM54 demonstrate antitumor activity in patient-derived xenograft models of soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1880.

Nicotine-derived nitrosamines promote renal cell carcinoma cell migration via binding to nicotinic acetylcholine receptors

Several risk factors such as smoking, obesity, diabetes, and hypertension are related to the incidence of kidney cancer. Renal cell carcinoma (RCC) originates from the renal tubular epithelium and is the major form of kidney cancer. Notably, the relative risk of RCC in smokers compared to non-smokers is 1.38 while revealing a dose-dependent risk reduction in smoking cessation. Carcinogenic substances in tobacco, specifically nicotine-derived nitrosamines like 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), were known to induce the formation of DNA adducts, mutating genes that potentially initiate cancers. In addition to carcinogenesis, nicotine-derived nitrosamines may also contribute to modify the malignant features, which accelerates the cancer progression. This study was aimed to evaluate the effects of NNK in the modulation RCC cell proliferation and motility, besides, the possible underlying mechanisms were also studied. Our findings showed differential expression of nicotinic acetylcholine receptor (nAChR) mRNA subunits between normal human renal tubular cell (HK-2) and RCC cells (Achn), suggesting that RCC cells may have the ability in respond to the stimuli of NNK. While NNK didn't significantly show an influence in RCC cell viability, it enhanced cell motility and migration ability. Utilizing specific inhibitors, it was inferred that NNK stimulates RCC cell migration via nAChR activation and subsequent calcium channel opening. Our research underscores the necessity of smoking cessation in RCC management, even as further studies on diverse RCC cell lines and real-world data are required.