Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-ß2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-ß2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

The ways and means that fine tune Sirt1 activity.

Sirt1 is the most evolutionarily conserved mammalian sirtuin. It plays a vital role in the regulation of metabolism, stress responses, genome stability, and ultimately aging. Although much attention has focused on the identification of the cellular targets and functional networks controlled by Sirt1, the mechanisms that regulate Sirt1 activity by biological stimuli have only recently begun to emerge. As an enzyme, the activity of Sirt1 can be controlled by the availability of its substrates, post-translational modifications, interactions with other proteins, or changes in its expression levels. In this review, we briefly discuss the ways and means by which the activity of Sirt1 is fine-tuned under different conditions.

Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine.

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC. Also it is known as an in vitro and in vivo mutagen and genotoxicant. However, the molecular mechanism by which procarbazine induces mutations is not thoroughly understood and the spectrum of procarbazine-induced in vivo mutations is described insufficiently. We employed flow cytometry-based erythrocyte and T lymphocyte assays in order to quantify the frequencies of cells deficient in glycosylphosphatidyl inositol-anchored surface markers CD59 and CD48 (presumed mutants in the endogenous X-linked Pig-a gene) in rats. The rats were treated once daily with 100 mg/kg procarbazine HCl for 3 days. In addition, we sorted mutant-phenotype spleen T cells and immediately analysed their Pig-a gene using next generation sequencing of dual-indexed multiplex libraries and error-correcting data filtering. More than 100-fold increase in the frequencies of CD59-deficient RBCs was observed at Day 29 after the last administration, and a 10-fold increase in the frequency of CD48-deficient T cells was observed at Days 45 to 50. Sequencing revealed that, in T cells from procarbazine-treated rats, mutations in the Pig-a gene occurred predominantly at A:T basepairs when A was located on the non-transcribed DNA strand. A→T transversion was the most common mutation. Our results suggest that, at least for the transcribed X-linked Pig-a gene, in vivo methyl guanine adducts are not the major contributors to mutations induced by procarbazine.

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene.

Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) compose the largest family of cell surface receptors and are the most common target of therapeutic drugs. The nonvisual arrestins, ß-arrestin-1 and ß-arrestin-2, are multifunctional scaffolding proteins that play critical roles in GPCR signaling. On binding of activated GPCRs at the plasma membrane, ß-arrestins terminate G protein-dependent responses (desensitization) and stimulate ß-arrestin-dependent signaling pathways. Alterations in the cellular complement of ß-arrestin-1 and ß-arrestin-2 occur in many human diseases, and their genetic ablation in mice has severe consequences. Surprisingly, however, the factors that control ß-arrestin gene expression are poorly understood. We demonstrate that glucocorticoids differentially regulate ß-arrestin-1 and ß-arrestin-2 gene expression in multiple cell types. Glucocorticoids act via the glucocorticoid receptor (GR) to induce the synthesis of ß-arrestin-1 and repress the expression of ß-arrestin-2. Glucocorticoid-dependent regulation involves the recruitment of ligand-activated glucocorticoid receptors to conserved and functional glucocorticoid response elements in intron-1 of the ß-arrestin-1 gene and intron-11 of the ß-arrestin-2 gene. In human lung adenocarcinoma cells, the increased expression of ß-arrestin-1 after glucocorticoid treatment impairs G protein-dependent activation of inositol phosphate signaling while enhancing ß-arrestin-1-dependent stimulation of the MAPK pathway by protease activated receptor 1. These studies demonstrate that glucocorticoids redirect the signaling profile of GPCRs via alterations in ß-arrestin gene expression, revealing a paradigm for cross-talk between nuclear and cell surface receptors and a mechanism by which glucocorticoids alter the clinical efficacy of GPCR-based drugs.

Assessment of in vivo chemical mutagenesis by long-read sequencing.

Evaluating the mutagenic properties of chemicals is crucial for understanding their potential cancer risks. Recent Illumina-based error-corrected sequencing techniques have enabled the direct detection of mutations induced de novo by mutagens. However, as the Illumina platform lacks intrinsic error-correction capabilities, complex library preparations and bioinformatic processes are necessary to identify these rare mutations. In this study, we evaluated whether long-read PacBio-based HiFi sequencing (HiFi seq), which has integrated error-correction, can detect de novo mutations induced by mutagens in C57BL/6 mouse tissues. Using HiFi seq, dose-dependent increases in mutation frequencies were found in tissues from mice exposed to 7,12-dimethylbenz[a]anthracene, procarbazine, and N-propyl-N-nitrosourea. Furthermore, the mutational signatures derived from these exposures were consistent with those previously reported for these mutagens. This study demonstrates that HiFi seq can complement established mutation detection assays to facilitate the identification of hazardous compounds.

Complete genome sequence of cephalosporin and tetracycline-resistant Citrobacter freundii CF51 isolate from a patient with urinary tract infection.

We report here the complete genome sequence of cephalosporin (cefazolin and cephalotin) and tetracycline-resistant Citrobacter freundii strain CF51, isolated from a patient with a urinary tract infection. The genome comprises a 5.0-Mb chromosome, along with an 87.2-kb plasmid and a 23.9-kb plasmid.

Whole-Genome Sequence Analysis of Antibiotic Resistance, Virulence, and Plasmid Dynamics in Multidrug-Resistant E. coli Isolates from Imported Shrimp.

We analyzed antimicrobial resistance and virulence traits in multidrug-resistant (MDR) E. coli isolates obtained from imported shrimp using whole-genome sequences (WGSs). Antibiotic resistance profiles were determined phenotypically. WGSs identified key characteristics, including their multilocus sequence type (MLST), serotype, virulence factors, antibiotic resistance genes, and mobile elements. Most of the isolates exhibited resistance to gentamicin, streptomycin, ampicillin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. Multilocus sequence type (MLST), serotype, average nucleotide identity (ANI), and pangenome analysis showed high genomic similarity among isolates, except for EC15 and ECV01. The EC119 plasmid contained a variety of efflux pump genes, including those encoding the acid resistance transcriptional activators (gadE, gadW, and gadX), resistance-nodulation-division-type efflux pumps (mdtE and mdtF), and a metabolite, H1 symporter (MHS) family major facilitator superfamily transporter (MNZ41_23075). Virulence genes displayed diversity, particularly EC15, whose plasmids carried genes for adherence (faeA and faeC-I), invasion (ipaH and virB), and capsule (caf1A and caf1M). This comprehensive analysis illuminates antimicrobial resistance, virulence, and plasmid dynamics in E. coli from imported shrimp and has profound implications for public health, emphasizing the need for continued surveillance and research into the evolution of these important bacterial pathogens.

Whole-genome high-fidelity sequencing: A novel approach to detecting and characterization of mutagenicity in vivo.

Direct DNA sequencing can be used for characterizing mutagenicity in simple and complex biological models. Recently we described a method of whole-genome sequencing for detecting mutations in simple models of cultured bacteria, mammalian cells, and nematode. In the current proof-of-concept study, we expand and improve our method for evaluating a more complex mammalian biological model in outbred mice. We detail the method by applying it to a small set of animals treated with a mutagen with known mutagenicity profiles, N-ethyl-N-nitrosourea (ENU), for consistency with the known data. Whole-genome high-fidelity sequencing (HiFi Sequencing) showed frequencies and spectra of background mutations in tissues of untreated mice that were consistent with normal ageing and characterized by spontaneous or enzymatic deamination of 5-methylcytosine. In mice treated with a single 40 mg/kg dose of ENU, the frequency of mutations in the genomic DNA of solid tissues increased up to 7-fold, with the greatest increase observed in the spleen and the smallest increase in the liver. The most common mutations detected in ENU-treated mice were T > A transitions and T > C transversions, consistent with the types of mutations caused by alkylating agents. The data suggest that HiFi Sequencing may be useful for characterizing mutagenicity of novel compounds in various biological models.

Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.

DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10-8 mutations per base pair. In this proof-of-principle study, we evaluated whether this technique could also detect the on- and off-target mutations generated by a cytosine-to-thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on-target mutant allele fractions ranging from ~7% to ~63%, depending on the single-guide RNA (sgRNA) used, while no on-target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3-fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on- and off-target mutations in cell populations that have undergone genome editing.

Draft Genome Sequences of 14 Fluoroquinolone-Resistant Escherichia coli Isolates from Imported Shrimp.

We report the draft genome sequences of 14 fluoroquinolone-resistant Escherichia coli strains that were isolated from imported shrimp. All isolates contained multiple point mutations in the quinolone resistance-determining regions (QRDRs) and non-QRDRs of gyrA, parC, and parE genes. The data improve the understanding of fluoroquinolone resistance and indicate resistance mechanisms.

Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme.

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.

Evaluation of the mutagenic effects of Molnupiravir and N4-hydroxycytidine in bacterial and mammalian cells by HiFi sequencing.

Molnupiravir (MOV) is used to treat COVID-19. In cells, MOV is converted to the ribonucleoside analog N4-hydroxycytidine (NHC) and incorporated into the SARS-CoV-2 RNA genome during its replication, resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS-CoV-2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow-frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E. coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome-wide mutation frequencies in a dose-dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.

Draft Genome Sequences of Tetracycline-Resistant Shiga Toxin-Producing Escherichia coli Isolates from Food.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen transmitted from animal to humans through contaminated food. Here, we report the draft genome sequences of six STEC isolates (six serotypes) from food (cheese, coriander, and pea protein pellets) in different countries; these isolates were resistant to tetracycline, with MIC values ranging from <1.5 to 256 µg/mL.

Genome-wide detection of ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing.

Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome-wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error-free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi). In this study, we show that PacBio HiFi can detect genome-wide ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms. The mutation frequencies (MFs) of unexposed samples in both models were ~1 × 10-7 mutations per base pair. Compared to these controls, PacBio HiFi detected MF increases of 23-fold in cultures of L5178Y cells exposed to 5 mM ethyl methanosulfonate (EMS) for 4 h, and 5-, 12-, and 29-fold in cultures of C. elegans worms exposed to 12.5, 25, and 50 mM EMS for 4 h, respectively. In both models, the mutation spectra of controls were diverse, while those derived from EMS-exposed samples were dominated by C:G → T:A transitions. To validate these results, clone sequencing analyses were performed on the same cultures of L5178Y cells. The results obtained by clone sequencing and PacBio HiFi were almost identical. Our results suggest that PacBio sequencing could be used for the detection, quantitation, and characterization of mutations in any DNA-containing sample, including those that are not compatible with conventional mutation detection approaches.

Structure of Nampt/PBEF/visfatin, a mammalian NAD+ biosynthetic enzyme.

Nicotinamide phosphoribosyltransferase (Nampt) synthesizes nicotinamide mononucleotide (NMN) from nicotinamide in a mammalian NAD+ biosynthetic pathway and is required for SirT1 activity in vivo. Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of NMN shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism.

PacBio sequencing detects genome-wide ultra-low-frequency substitution mutations resulting from exposure to chemical mutagens.

Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, and often require specific cell lines or animal models-features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single-Molecule, Real-Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error-free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N-ethyl-N-nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone-sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10-7 mutations per base pair (mut/bp).

Mutational signatures in T-lymphocytes of rats treated with N-propyl-N-nitrosourea and procarbazine.

We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU-induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ-induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10-40× increases in the number of mutations per clone were detected in T-cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X-linked Pig-a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.