Biology subjects

Engelward, B. P.

Publications and source records attributed to Engelward, B. P..

5 recordsLinked to original sources

bioRxiv2025

DNA adduct and mutational profiles reveal a threshold of cellular defenses against N-nitrosodimethylamine administered to mice in drinking water

N-Nitrosodimethylamine (NDMA) is classified as an animal and probable human carcinogen. Murine liver DNA adducts, mutations, MGMT and CYP2E1 were evaluated following chronic administration of NDMA in drinking water. In a dose-escalation study, 7-methylguanine (m7G) increased linearly with NDMA dose. O6-Methylguanine (m6G) remained near background for NDMA doses up to [~]1 ppm, beyond which its level, and corresponding mutations, rose steeply. An extended study was done with 5 ppm NDMA, in which adducts were measured at 3 weeks and mutations at 10 weeks. While the level of CYP2E1 was unchanged, MGMT gene transcription was induced in females at 10 weeks. Homologous recombination-mediated chromosomal rearrangements did not increase over background. Point mutations, however, were elevated substantially in both sexes. Mutational analysis over 96 trinucleotide contexts revealed predominantly GC[->]AT mutations in 5-purine-G-3 contexts in a pattern matching human COSMIC cancer mutational signature SBS11, with secondary features resembling SBS119 (AT[->]GC). Taken together, the data implicate m6G as the dominant mutagenic adduct under chronic dosing with NDMA. Furthermore, the genomic m6G level was identified at which its dedicated repair protein, MGMT, became saturated. The coordinated application of DNA adduct, mutational and biochemical analyses provides a new approach for early detection and cancer management. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/687536v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e6ae72org.highwire.dtl.DTLVardef@1c6fad6org.highwire.dtl.DTLVardef@7e3716org.highwire.dtl.DTLVardef@52a87b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Persistent interferon signaling and clonal expansion mark early events in DNA methylation damage-induced liver cancer

N-Nitrosodimethylamine (NDMA), a probable human carcinogen, induces toxic and mutagenic O6-methylguanine lesions that are repaired by the O6-methylguanine methyltransferase (MGMT). To elucidate mechanisms of NDMA-induced liver cancer progression, we performed longitudinal analyses of phenomic, transcriptomic, and phosphoproteomic changes in wild-type and MGMT-deficient mice, observing amplified responses in the deficient genotype. Early molecular rewiring indicative of a DNA damage response was detected by phosphoproteomic and transcriptomic profiling within days post-exposure. Transcriptomic analyses identified a persistent and robust interferon response as the dominant activated pathway. This chronic interferon signaling, which remained unresolved, correlated with extensive clonal expansion, an early hallmark of oncogenesis. Spatial transcriptomics further revealed pathway alterations favoring tumorigenesis within clonally expanded cells. These findings delineate the cascade of molecular events triggered by acute early-life NDMA exposure, culminating in cancer development months later. Our study unveils potential predictive biomarkers and strategies for disease mitigation.

cancer biology↗

bioRxiv2025

Sex and Alkyladenine DNA Glycosylase Expression are Key Susceptibility Factors for NDMA-induced Mutations, Toxicity, and Cancer

N-Nitrosodimethylamine (NDMA) is present in food, water, and drugs and is considered a probable human carcinogen by the International Agency for Research on Cancer. The mechanism of action of NDMA involves the generation of carcinogenic methyl lesions such as 3-methyladenine (3MeA) on DNA bases. Alkyladenine DNA Glycosylase (AAG) removes 3MeA to initiate Base Excision Repair, leaving an intermediary lesion that is subsequently resolved by backbone cleavage, nucleotide insertion, and backbone ligation. The intermediate steps following lesion removal produce potentially toxic and mutagenic single-strand DNA breaks. Here, we explored differences between males and females regarding downstream DNA damage, toxicity, mutations and cancer arising from 3MeA in the livers of WT, Aag-/-, and Aag-overexpressing (AagTg) mice. We found that males were more susceptible to NDMA-induced mutations (WT and Aag-/-) and cancer (all genotypes). In contrast, AagTg females were more prone than males to micronucleus induction. As we showed in our prior analyses where data were pooled for males and females, Aag-/- mice were significantly more susceptible to NDMA-induced mutations and cancer, and AagTg mice displayed significantly greater toxicity. Building on these findings, our analyses of sex-related differences show that Aag deficiency and maleness are both susceptibility factors for NDMA-induced liver cancer, while Aag overexpression drives toxicity, potentially with a greater effect on females. By assessing differences between males and females, this study reveals a deeper mechanistic understanding of the underpinnings for a well-known increased risk of liver cancer in men versus women by demonstrating a higher susceptibility of male mice to both mutations and cancer.

cancer biology↗

bioRxiv2021

CometChip Enables Parallel Analysis of Multiple DNA Repair Activities

DNA damage can be cytotoxic and mutagenic and is directly linked to aging, cancer, and heritable diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these advanced applications of the CometChip platform, we expand the efficacy of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

molecular biology↗

bioRxiv2021

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice

N-nitrosodimethylamine (NDMA) is a DNA methylating agent that has been discovered to contaminate water, food and drugs. The alkyladenine glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we studied Aag-/- and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine, or 3MeA) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switched from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrated a suite of molecular, cellular and physiological analyses. We found that unrepaired 3MeA is somewhat toxic but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tips the balance between blocks and breaks, and thus dictates the disease consequences of DNA damage.

cancer biology↗