PARP14 is regulated by the PARP9/DTX3L complex and promotes interferon γ-induced ADP-ribosylation.

Protein ADP-ribosylation plays important but ill-defined roles in antiviral signalling cascades such as the interferon response. Several viruses of clinical interest, including coronaviruses, express hydrolases that reverse ADP-ribosylation catalysed by host enzymes, suggesting an important role for this modification in host-pathogen interactions. However, which ADP-ribosyltransferases mediate host ADP-ribosylation, what proteins and pathways they target and how these modifications affect viral infection and pathogenesis is currently unclear. Here we show that host ADP-ribosyltransferase activity induced by IFNγ signalling depends on PARP14 catalytic activity and that the PARP9/DTX3L complex is required to uphold PARP14 protein levels via post-translational mechanisms. Both the PARP9/DTX3L complex and PARP14 localise to IFNγ-induced cytoplasmic inclusions containing ADP-ribosylated proteins, and both PARP14 itself and DTX3L are likely targets of PARP14 ADP-ribosylation. We provide evidence that these modifications are hydrolysed by the SARS-CoV-2 Nsp3 macrodomain, shedding light on the intricate cross-regulation between IFN-induced ADP-ribosyltransferases and the potential roles of the coronavirus macrodomain in counteracting their activity.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

Tissue Specificity of DNA Damage and Repair.

DNA is a remarkable biochemical macromolecule tasked with storing the genetic information that instructs life on planet Earth. However, its inherent chemical instability within the cellular milieu is incompatible with the accurate transmission of genetic information to subsequent generations. Therefore, biochemical pathways that continuously survey and repair DNA are essential to sustain life, and the fundamental mechanisms by which different DNA lesions are repaired have remained well conserved throughout evolution. Nonetheless, the emergence of multicellular organisms led to profound differences in cellular context and physiology, leading to large variations in the predominant sources of DNA damage between different cell types and in the relative contribution of different DNA repair pathways toward genome maintenance in different tissues. While we continue to make large strides into understanding how individual DNA repair mechanisms operate on a molecular level, much less attention is given to these cell type-specific differences. This short review aims to provide a broad overview of DNA damage and repair mechanisms to nonspecialists and to highlight some fundamental open questions in tissue and cell-type-specificity of these processes, which may have profound implications for our understanding of important pathophysiological processes such as cancer, neurodegeneration, and aging.

Biallelic variants in DNA2 cause poikiloderma with congenital cataracts and severe growth failure reminiscent of Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare, heterogeneous autosomal recessive genodermatosis, with poikiloderma as its hallmark. It is classified into two types: type I, with biallelic variants in ANAPC1 and juvenile cataracts, and type II, with biallelic variants in RECQL4, increased cancer risk and no cataracts. We report on six Brazilian probands and two siblings of Swiss/Portuguese ancestry presenting with severe short stature, widespread poikiloderma and congenital ocular anomalies. Genomic and functional analysis revealed compound heterozygosis for a deep intronic splicing variant in trans with loss of function variants in DNA2, with reduction of the protein levels and impaired DNA double-strand break repair. The intronic variant is shared by all patients, as well as the Portuguese father of the European siblings, indicating a probable founder effect. Biallelic variants in DNA2 were previously associated with microcephalic osteodysplastic primordial dwarfism. Although the individuals reported here present a similar growth pattern, the presence of poikiloderma and ocular anomalies is unique. Thus, we have broadened the phenotypical spectrum of DNA2 mutations, incorporating clinical characteristics of RTS. Although a clear genotype-phenotype correlation cannot be definitively established at this moment, we speculate that the residual activity of the splicing variant allele could be responsible for the distinct manifestations of DNA2-related syndromes.

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia.

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling.

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.

Dynll1 is essential for development and promotes endochondral bone formation by regulating intraflagellar dynein function in primary cilia.

Mutations in subunits of the cilia-specific cytoplasmic dynein-2 (CD2) complex cause short-rib thoracic dystrophy syndromes (SRTDs), characterized by impaired bone growth and life-threatening perinatal respiratory complications. Different SRTD mutations result in varying disease severities. It remains unresolved whether this reflects the extent of retained hypomorphic protein functions or relative importance of the affected subunits for the activity of the CD2 holoenzyme. To define the contribution of the LC8-type dynein light chain subunit to the CD2 complex, we have generated Dynll1-deficient mouse strains, including the first-ever conditional knockout (KO) mutant for any CD2 subunit. Germline Dynll1 KO mice exhibit a severe ciliopathy-like phenotype similar to mice lacking another CD2 subunit, Dync2li1. Limb mesoderm-specific loss of Dynll1 results in severe bone shortening similar to human SRTD patients. Mechanistically, loss of Dynll1 leads to a partial depletion of other SRTD-related CD2 subunits, severely impaired retrograde intra-flagellar transport, significant thickening of primary cilia and cilia signaling defects. Interestingly, phenotypes of Dynll1-deficient mice are very similar to entirely cilia-deficient Kif3a/Ift88-null mice, except that they never present with polydactyly and retain relatively higher signaling outputs in parts of the hedgehog pathway. Compared to complete loss of Dynll1, maintaining very low DYNLL1 levels in mice lacking the Dynll1-transcription factor ASCIZ (ATMIN) results in significantly attenuated phenotypes and improved CD2 protein levels. The results suggest that primary cilia can maintain some functionality in the absence of intact CD2 complexes and provide a viable animal model for the analysis of the underlying bone development defects of SRTDs.

Host ADP-ribosylation and the SARS-CoV-2 macrodomain.

The COVID-19 pandemic has prompted intense research efforts into elucidating mechanisms of coronavirus pathogenesis and to propose antiviral interventions. The interferon (IFN) response is the main antiviral component of human innate immunity and is actively suppressed by several non-structural SARS-CoV-2 proteins, allowing viral replication within human cells. Differences in IFN signalling efficiency and timing have emerged as central determinants of the variability of COVID-19 disease severity between patients, highlighting the need for an improved understanding of host-pathogen interactions that affect the IFN response. ADP-ribosylation is an underexplored post-translational modification catalyzed by ADP-ribosyl transferases collectively termed poly(ADP-ribose) polymerases (PARPs). Several human PARPs are induced by the IFN response and participate in antiviral defences by regulating IFN signalling itself, modulating host processes such as translation and protein trafficking, as well as directly modifying and inhibiting viral target proteins. SARS-CoV-2 and other viruses encode a macrodomain that hydrolyzes ADP-ribose modifications, thus counteracting antiviral PARP activity. This mini-review provides a brief overview of the known targets of IFN-induced ADP-ribosylation and the functions of viral macrodomains, highlighting several open questions in the field.

ADP-ribosylation: from molecular mechanisms to human disease.

Post-translational modification of proteins by ADP-ribosylation, catalysed by poly (ADP-ribose) polymerases (PARPs) using NAD+ as a substrate, plays central roles in DNA damage signalling and repair, modulates a range of cellular signalling cascades and initiates programmed cell death by parthanatos. Here, we present mechanistic aspects of ADP-ribose modification, PARP activation and the cellular functions of ADP-ribose signalling, and discuss how this knowledge is uncovering therapeutic avenues for the treatment of increasingly prevalent human diseases such as cancer, ischaemic damage and neurodegeneration.

The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair.

The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.

Use of quantitative mass spectrometric analysis to elucidate the mechanisms of phospho-priming and auto-activation of the checkpoint kinase Rad53 in vivo.

The cell cycle checkpoint kinases play central roles in the genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, the mechanisms by which these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we used quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and we show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1 target site phosphorylation patterns of Rad53, including previously undetected tri- and tetraphosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.

Synthesis of Structural ADP-Ribose Analogues as Inhibitors for SARS-CoV-2 Macrodomain 1.

Protein adenosine diphosphate (ADP)-ribosylation is crucial for a proper immune response. Accordingly, viruses have evolved ADP-ribosyl hydrolases to remove these modifications, a prominent example being the SARS-CoV-2 NSP3 macrodomain, "Mac1". Consequently, inhibitors are developed by testing large libraries of small molecule candidates, with considerable success. However, a relatively underexplored angle in design pertains to the synthesis of structural substrate mimics. Here, we present the synthesis and biophysical activity of novel adenosine diphosphate ribose (ADPr) analogues as SARS-CoV-2 NSP3 Mac1 inhibitors.

Targeting Myeloperoxidase Ameliorates Gouty Arthritis: A Virtual Screening Success Story.

This study presents a new approach for identifying myeloperoxidase (MPO) inhibitors with strong in vivo efficacy. By combining inhibitor-like rules and structure-based virtual screening, the pipeline achieved a 70% success rate in discovering diverse, nanomolar-potency reversible inhibitors and hypochlorous acid (HOCl) scavengers. Mechanistic analysis identified RL6 as a genuine MPO inhibitor and RL7 as a potent HOCl scavenger. Both compounds effectively suppressed HOCl production in cells and neutrophils, with RL6 showing a superior inhibition of neutrophil extracellular trap release (NETosis). In a gout arthritis mouse model, intraperitoneal RL6 administration reduced edema, peroxidase activity, and IL-1ß levels. RL6 also exhibited oral bioavailability, significantly reducing paw edema when administered orally. This study highlights the efficacy of integrating diverse screening methods to enhance virtual screening success, validating the anti-inflammatory potential of potent inhibitors, and advancing the MPO inhibitor research.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

ATM substrate Chk2-interacting Zn2+ finger (ASCIZ) Is a bi-functional transcriptional activator and feedback sensor in the regulation of dynein light chain (DYNLL1) expression.

The highly conserved DYNLL1 (LC8) protein was originally discovered as a light chain of the dynein motor complex, but is increasingly emerging as a sequence-specific regulator of protein dimerization with hundreds of targets and wide-ranging cellular functions. Despite its important roles, DYNLL1's own regulation remains poorly understood. Here we identify ASCIZ (ATMIN/ZNF822), an essential Zn(2+) finger protein with dual roles in the DNA base damage response and as a developmental transcription factor, as a conserved regulator of Dynll1 gene expression. DYNLL1 levels are reduced by ∼10-fold in the absence of ASCIZ in human, mouse and chicken cells. ASCIZ binds directly to the Dynll1 promoter and regulates its activity in a Zn(2+) finger-dependent manner. DYNLL1 protein in turn interacts with ten binding sites in the ASCIZ transcription activation domain, and high DYNLL1 levels inhibit the transcriptional activity of ASCIZ. In addition, DYNLL1 was also required for DNA damage-induced ASCIZ focus formation. The dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. The ASCIZ-DYNLL1 feedback loop represents a novel mechanism for auto-regulation of gene expression, where the gene product directly inhibits the transcriptional activator while bound at its own promoter.

Rothmund-Thomson syndrome, a disorder far from solved.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by a range of clinical symptoms, including poikiloderma, juvenile cataracts, short stature, sparse hair, eyebrows/eyelashes, nail dysplasia, and skeletal abnormalities. While classically associated with mutations in the RECQL4 gene, which encodes a DNA helicase involved in DNA replication and repair, three additional genes have been recently identified in RTS: ANAPC1, encoding a subunit of the APC/C complex; DNA2, which encodes a nuclease/helicase involved in DNA repair; and CRIPT, encoding a poorly characterized protein implicated in excitatory synapse formation and splicing. Here, we review the clinical spectrum of RTS patients, analyze the genetic basis of the disease, and discuss molecular functions of the affected genes, drawing some novel genotype-phenotype correlations and proposing avenues for future studies into this enigmatic disorder.

DNA polymerase eta protects human cells against DNA damage induced by the tumor chemotherapeutic temozolomide.

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

DNA damage in tissues and organs of mice treated with diphenyl diselenide.

Diphenyl diselenide (DPDS) is an organoselenium compound with interesting pharmacological activities and various toxic effects. In previous reports, we demonstrated the pro-oxidant action and the mutagenic properties of this molecule in bacteria, yeast and cultured mammalian cells. This study investigated the genotoxic effects of DPDS in multiple organs (brain, kidney, liver, spleen, testes and urinary bladder) and tissues (bone marrow, lymphocytes) of mice using in vivo comet assay, in order to determine the threshold of dose at which it has beneficial or toxic effects. We assessed the mechanism underlying the genotoxicity through the measurement of GSH content and thiobarbituric acid reactive species, two oxidative stress biomarkers. Male CF-1 mice were given 0.2-200 micromol/kg BW DPDS intraperitonially. DPDS induced DNA damage in brain, liver, kidney and testes in a dose response manner, in a broad dose range at 75-200 micromol/kg with the brain showing the highest level of damage. Overall, our analysis demonstrated a high correlation among decreased levels of GSH content and an increase in lipid peroxidation and DNA damage. This finding establishes an interrelationship between pro-oxidant and genotoxic effects. In addition, DPDS was not genotoxic and did not increase lipid peroxidation levels in any organs at doses < 50 micromol/kg. Finally, pre-treatment with N-acetyl-cysteine completely prevented DPDS-induced oxidative damage by the maintenance of cellular GSH levels, reinforcing the positive relationship of DPDS-induced GSH depletion and DNA damage. In summary, DPDS induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney and testes.