Obesity is a major global public health challenge associated with chronic low-grade inflammation and oxidative stress. Although natural antioxidant compounds have been investigated to mitigate obesity-related molecular damage, the effects of bergamot (Citrus bergamia) leaf extract (BLE) on DNA integrity remains unclear. This study evaluated the effects of BLE on hepatic DNA damage in diet-induced obese rats. Forty Wistar rats were fed either a control or a high-sugar-fat (HSF) diet and treated with BLE by oral gavage. The adiposity index was recorded for all groups, and hepatic DNA damage was assessed using the comet assay. The HSF diet significantly increased both the adiposity index and DNA strand breaks in the liver compared with the control group. Notably, BLE treatment was associated with a lower adiposity index and reduced hepatic DNA strand breaks measured by the comet assay in obese rats (p < 0.05). These findings indicate an association between BLE treatment and reduced hepatic DNA damage in this model of diet-induced obesity.
Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) have transformed precision oncology by exploiting synthetic lethality in homologous recombination (HR)-deficient cancers, with multiple FDA-approved agents targeting BRCA1/2-mutant tumors. Despite initial efficacy, resistance inevitably emerges, limiting long-term clinical benefit. This review synthesizes emerging mechanistic insights into PARPi response and resistance. Recent evidence reframes PARP inhibition cytotoxicity through a transcription-replication conflict model and identifies single-stranded DNA gaps as the primary lethal lesion in HR-deficient cells, rather than double-strand breaks. These findings suggest that resistance reflects restoration of replication gap suppression or resolution of transcription-replication stress. We further highlight DNA ligase III as a collateral vulnerability in 53BP1-deficient resistant tumors, and discuss proteolysis-targeting chimera (PROTAC)-based PARP1 degraders as a strategy to overcome resistance and induce alternative cell death pathways. Established resistance mechanisms-including BRCA1/2 reversion mutations, shieldin complex loss, RAD51 hyperactivation, and pharmacokinetic alterations-are reconsidered within this updated framework. Combination strategies with ATR inhibitors show promising clinical activity in PARPi-resistant HR-deficient ovarian cancer. Finally, we propose an integrated biomarker framework combining HRD scar assays, functional RAD51 foci analysis, replication gap profiling, and circulating tumor DNA (ctDNA) monitoring to enable dynamic resistance tracking.
DNA damage is ubiquitous and can arise from numerous sources. To mitigate the potential effects of DNA damage, cells possess varied DNA repair and damage tolerance mechanisms. Conserved throughout evolution, specialized DNA damage-bypass DNA polymerases from the Y family provide DNA damage tolerance. E. coli harbors two Y-family polymerases whereas humans have four members of the Y family. E. coli DinB and human DNA polymerase kappa have shown similar damage bypass profiles in that they both are specific for minor groove adducts and are inhibited by major groove adducts. These two proteins share a similar active site loop that is adjacent to the nascent base pair. These active site loops were analyzed by alanine scanning mutagenesis with the resulting proteins characterized in primer extension assays and for their thermal stability. Most variants show similar activity to the respective wild-type proteins, with a few mutations resulting in dramatic losses of activity and changes in stability. The effects of the mutations are remarkably similar in DinB and polymerase kappa, with mutation of specific aligned residues showing decreased activity and/or stability. Most of the variants have similar thermal stability as the respective wild-type proteins and show the characteristic increase in stability in the presence of substrates, with the less active variants in general showing limited stabilization by DNA or DNA and incoming nucleotides.
Ethylene oxide (EtO) is primarily used as an intermediate in the manufacture of chemicals, with a minor use as a sterilant for medical equipment and food products. It is a direct-acting alkylating agent that reacts with cellular macromolecules, including proteins and DNA. EtO has been shown to induce tumors in rodents and humans. DNA reactivity has been the postulated mode of action (MOA) for its carcinogenicity. The current study has investigated the dose response for EtO-induced genetic damage to inform the biological plausibility of a dose-response model for cancer risk assessment. Male and female B6C3F1 mice (≥ 10/sex/concentration) were exposed to 0, 0.05, 0.1, 0.5, 1, 50, 100, or 200 ppm EtO by whole-body inhalation (6 h/day for 28 days, 7 days/week). Mutagenicity was assessed by determining the frequency of mutant Pig-a phenotype in reticulocytes (RET) and mature red blood cells (RBC) on Day 28. Cytogenetic damage was evaluated by the erythrocyte micronucleus (MN) test in blood samples collected on Days 5 and 28. EtO is a relatively weak genotoxicant with treatment-related increases in Pig-a and MN frequencies being seen primarily at 200 ppm. The hockey-stick shaped dose response for genetic damage may be conservatively interpreted as being no more than a linear response with a single slope. Thus, a cancer risk assessment dose-response model consisting of a single shallow linear slope throughout the exposure range is biologically plausible and consistent if EtO were acting through a mutagenic MoA for its carcinogenicity.
There is growing interest in in vitro-based new approach methodologies (NAMs) for assessing genotoxicity. These approaches are desirable for industries like cosmetics which are prohibited from animal testing due to regulatory restrictions, as well as "3R" considerations to avoid in vivo testing. One NAM is the adaptation of the in vitro micronucleus assay for metabolically competent human hepatic HepaRG cells. However, per OECD Test Guideline 487, the use of non-validated cell types such as these needs to be justified based on their proficiency in the assay. To this end, we have tested 28 chemicals in HepaRG and TK6 human lymphoblast cells, a standard cell line considered validated for use in the in vitro micronucleus assay. HepaRG cells showed a high overall accuracy of 86%, compared to 75% for TK6 cells across a balanced set of expected in vivo positives and negatives across various genotoxic modes of action. The improved overall performance of HepaRG cells was predominantly based on their lower susceptibility towards compounds known to cause "false positive" responses (positive results in vitro, but are known to be negative in vivo), particularly those related to oxidative stress as demonstrated by the ToxTracker assay. The study indicates a high predictive capacity of HepaRG cells, suggesting that with further examination of chemicals requiring metabolic activation, they may be suitable for regulatory testing. These findings advocate for the incorporation of HepaRG cells into genotoxicity assessment frameworks, providing a more human-relevant approach that has the potential to improve risk assessment outcomes.
Haloacetaldehyde disinfection by-products (HAL-DBPs), a class of unregulated emerging contaminants formed during drinking water chlorination, are widely detected in the aquatic environment, yet their potential reproductive toxicity remains poorly understood. In this study, we used a chronic drinking water exposure model in mice (90 days) and subsequently evaluated oocyte developmental competence in vitro. A chronic drinking water exposure model with environmentally relevant concentrations of HAL-DBPs was established to systematically evaluate their effects on oocyte developmental competence and to investigate the protective role of the natural antioxidant resveratrol (RES). The results showed that HAL-DBPs exposure significantly reduced oocyte maturation rate and early embryonic development potential, leading to spindle disorganization, chromosomal misalignment, and disruption of the actin cytoskeleton. Moreover, HAL-DBPs induced mitochondrial membrane potential loss, lipid metabolic disorder, impaired autophagy-lysosomal flux, excessive ROS accumulation, DNA damage, and apoptosis. Notably, RES cotreatment markedly alleviated these adverse effects by restoring mitochondrial function and redox homeostasis, reducing DNA damage and apoptosis, and preserving cytoskeletal integrity. Collectively, this study provides the first evidence that HAL-DBPs directly impair female germ cells through mitochondria-dependent oxidative stress mechanisms and proposes RES as a promising natural antioxidant intervention to mitigate reproductive toxicity associated with drinking water disinfection by-products.
While commonly described as an inhibitor of the histone methyltransferase EZH2, the compound 3-deazaneplanocin A (DZNep) functions as an inhibitor of S-adenosylhomocysteine hydrolase (SAHH), leading to pan-methyltransferase inhibition affecting over 150 individual enzyme targets. DZNep has been associated with significant benefits in limiting the development of multiple diseases in animal models. This review synthesizes evidence from preclinical studies demonstrating DZNep's therapeutic potential across multiple disease contexts. The compound shows particular promise as an anticancer and antifibrotic agent. DZNep exhibits consistent anti-inflammatory effects across diverse disease models. The compound shows a favorable safety profile at therapeutic doses, though comprehensive toxicology data remain limited to short-term animal studies. While some effects correlate with EZH2 inhibition, the breadth of DZNep's therapeutic activity suggests mechanisms extending beyond this single target. Clinical translation remains the critical next step for validating DZNep's potential and safety as a multi-target therapeutic agent capable of mitigating diverse pathological processes through methyltransferase modulation.
Polycyclic aromatic hydrocarbons (PAHs) are well-known for their mutagenic and carcinogenic effects. Benzo[b]fluoranthene (BbF) is one of 16 PAHs prioritized by the US Environmental Protection Agency for toxicological evaluation due to pervasive human exposure. As part of a multi-stakeholder consortium, the genotoxic effects of BbF were evaluated in MutaMouse males exposed to five doses of BbF or a vehicle control via repeated oral gavage for 28, 60, 90, 120, or 180 days, with dose ranges adjusted by duration of exposure. Mutagenesis was evaluated in lung tissue (n = 4) at 28, 90, and 180 days using Duplex Sequencing (DS), and chromosomal damage (n = 8) was evaluated using the micronucleus assay in peripheral blood at all time points. Dose- and time-dependent increases in total mutation frequency (MF) and C:G > A:T mutations were observed in lung tissue after 28, 90, and 180 days of exposure. By 28 days, BbF exposure produced lung cancer-associated mutational signatures linked to tobacco smoking. Mutations accumulated over time in lung, whereas chromosomal damage in peripheral blood erythrocytes reached a steady state by 28 days. Benchmark dose (BMD) confidence intervals (CIs) narrowed with extended exposure only for MF. Collectively, the data demonstrate that BbF is a potent mutagen capable of inducing cancer-relevant mutations in lung, supporting its potential role in human lung carcinogenesis. By distinguishing early mutagenic responses from cumulative mutation effects over time, these findings highlight the value of integrating mutagenicity assessment into extended-duration studies to better inform the potential health effects of chronic genotoxic exposures.
The adverse outcome pathway (AOP) framework has emerged as an important tool in mechanistic toxicology, providing chemically agnostic representations of the causal biological sequence from molecular initiating events (MIEs) to apical adverse outcomes (AOs). Yet, traditional AOP construction has relied predominantly on siloed, single-layer biological data, limiting both the mechanistic resolution and quantitative utility of AOPs in regulatory risk assessment. The proliferation of multi-omics technologies, such as transcriptomics, proteomics, metabolomics, and epigenomics, and their single-cell counterparts, now offers new opportunities to populate, validate, and quantify AOP networks with rich, multi-scale molecular data. This review systematically examines how each omics layer contributes uniquely to AOP development and discusses emerging frameworks for their integration. We describe the mechanistic logic underpinning transcriptome-guided key event (KE) identification, proteomic confirmation of KE-to-KE relationships (KERs), metabolomics-based linkage to phenotypic outcomes, epigenomic annotation of persistent and transgenerational effects, and single-cell resolution approaches that dissolve the cell population averaging problem inherent in bulk assays. We further assess quantitative AOP (qAOP) strategies built on benchmark dose (BMD) modeling of omics data, with emerging evidence that transcriptomic points of departure (tPODs) derived from short-term exposures are concordant with chronic apical endpoints. Critical knowledge gaps are identified, including incomplete molecular annotation of KEs in AOP-Wiki, the absence of standardized multiomics bioinformatics pipelines, the underdevelopment of epigenomic and spatial transcriptomic AOP layers, and regulatory hurdles impeding the translation of omics-derived PODs into health-based guidance values (HBGVs). We conclude with a forward-looking framework and research priorities to accelerate the regulatory acceptance of multiomics-informed AOPs as tools for next-generation chemical risk assessment.
Arsenic poisoning significantly elevates the risk of cancer and other chronic illnesses. The goal of this research is to identify important genes whose expression changes in response to arsenic toxicity, and the molecular pathways affected by arsenic, using computational analysis of arsenic toxicity profiles. This approach will computationally identify and analyze genes whose expression changes in response to arsenic, thereby elucidating the heightened risk of carcinogenesis in arsenic-exposed individuals. This work employed high-throughput arsenic toxicity profiles to computationally identify and analyze expressed genes (DEGs) differentially in Affymetrix microarray datasets from the Gene Expression Omnibus (GEO) database, which were screened using the GEO2R program. A protein-protein interaction (PPI) network was constructed using STRING to elucidate the functional links between these DEGs and DNA repair genes. Interactions between the seven central genes (E2F1, EXO1, EZH2, FEN1, HIST1H3A, POLA1, and TIMELESS) and the repair genes PARP1, NBN, PMS1, MSH3, XRCC5, XRCC6, MGMT, and MLH1 were discovered. We employed the DAVID and Enrichr-KG platforms to investigate the functions of these genes and their associations with cellular and molecular processes in greater detail. Two hundred eighty-one non-synonymous single-nucleotide polymorphisms (nsSNPs) in the 07 genes linked to arsenic toxicity were found using the COSMIC database. Based on our analysis, mutations in E2F1, EXO1, EZH2, FEN1, HIST1H3A, POLA1, and TIMELESS can hinder DNA repair mechanisms, ultimately leading to cancer. Our computational analysis demonstrated that these non-synonymous SNPs can affect gene function, potentially altering protein stability and activity. Furthermore, according to Metal-Protein docking and protein-protein docking, these genes and their mutations appear to affect interactions with repair proteins substantially. Specific dietary consumption may lessen the detrimental effects of arsenic poisoning on protein function. We hypothesized that the mutations might be reversed by attaching particular molecules to these mutants. The protective effects of six curcumin compounds were examined using molecular docking with AutoDock 4.2.6 to assess protein dynamics and binding interactions. Optimal complexes were selected for dynamics simulation using GROMACS, and potential strategies for long-term cancer prevention related to arsenic exposure were identified.
Epigenetic mechanisms regulating DNA gene expression have gained significant attention in recent years. MicroRNAs (miRNAs) play a key role in these mechanisms by modulating gene activity and relaying information throughout the body. This study investigates the potential of a radioprotective homeopathic solution, ultra-diluted Cadmium Sulfuratum (UCS), to mitigate the acute side effects of radiotherapy by modulating miRNA expression. Radiotherapy, a crucial cancer treatment, unavoidably exposes normal tissues to radiation, often causing acute side effects such as bone marrow suppression and enteritis, which can lead to severe clinical conditions. Emerging evidence suggests that miRNAs can serve as biomarkers for predicting radiation-induced side effects before histopathological changes occur. This study investigated the histopathological effects of UCS on intestinal tissues and its impact on serum miRNA expression in Swiss albino mice exposed to total body irradiation (TBI) at 8 Gy. Specifically, seven miRNAs previously validated as biodosimeters were analyzed using quantitative PCR (qPCR) on the 2nd and sixth days post-irradiation. Additionally, histopathological and immunohistochemical analyses were conducted on intestinal epithelial cells. Our results revealed that UCS application significantly altered the expression levels of miRNAs, including mmu-miR-150-5p, mmu-mir-320a-5p, mmu-mir-200b-5p, mmu-mir-30a-5p, and mmu-miR-29a-5p, compared to radiation-only groups. Histopathological evaluations demonstrated that UCS reduced radiation-induced damage to intestinal epithelial cells. In conclusion, UCS exhibited radioprotective properties by modulating miRNA expression and alleviating acute radiation-induced intestinal damage. These findings highlight the potential of UCS in epigenetic modulation and its application in mitigating the side effects of radiotherapy, providing a foundation for future clinical research.
Exposure to air pollution is associated with adverse health effects such as asthma, lung cancer, and cardiovascular diseases. Children are especially vulnerable due to their developing organs, immature immune systems, and higher ventilation rates relative to their body size. Although the adverse health effects of air pollution are well established, the contribution of specific pollutant constituents and the biological mechanisms underlying these effects are not fully understood. This pilot study investigates biomarkers of oxidative stress and inflammation and their association with urinary biomarkers of air pollutant exposure in children. Eighteen children were recruited from an elementary school in Stockton, CA and provided a total of 67 urine samples. Biomarkers of oxidative stress and inflammation were measured using enzyme-linked immunosorbent assays. Liquid chromatography with tandem mass spectrometry was used to measure urinary metabolites of six VOCs and four PAHs. Biomarkers of oxidative stress and inflammation were positively correlated with urinary metabolites of acrylonitrile, acrolein, crotonaldehyde, naphthalene, and fluorene. Two-fold increases in crotonaldehyde and naphthalene metabolite levels were significantly associated with 8-Isop, 8-OHdG and PGE2, with percent-changes ranging from 13%-21% and 11%-16%, respectively. A two-fold increase in crotonaldehyde metabolite level was also significantly associated with a 41% change in CC16. These results suggest that urinary biomarkers of oxidative stress and inflammation may serve as useful tools for assessing chemical exposures and early biological effects in children.
We demonstrate the utility of the tdk reporter gene system, by showing its ability to not only analyze mutational hotspots, but also to allow the analysis of both weak and strong mutator or mutagen effects. It can detect large and small insertions and deletions, as well as base substitutions. We previously defined cisplatin (CPT)-induced hotspots in tdk. One extraordinary hotspot for G:C- > T:A transversions at base pair (bp) 499 is in a region of the gene that appears to be mutationally prone, suggesting that the conformation of the DNA in that region may play a major role in elevating mutation rates. Here, we examined CPT-induced mutations in a derivative of the starting wild-type strain that lacks the nucleoid binding protein HNS, which is involved in the folding and compaction of the E. coli chromosome. In this strain background the extraordinary CPT-induced hotspot disappears, pinpointing the importance of DNA conformation in mutation rates at certain positions. We further show that this A:T-rich gene is a magnet for transposable elements.
Copper pyrithione (CuPT), a widely used antimicrobial agent in antifouling coatings and consumer products, has potential reproductive toxicity. This study aims to evaluate the impact of CuPT on human trophoblast-like JEG-3 cells and elucidate potential molecular mechanisms. Proliferation was assessed by CCK-8; migration and invasion by wound-healing and Matrigel Transwell assays; cell cycle and apoptosis by flow cytometry; intracellular reactive oxygen species (ROS) by DCFH-DA; DNA double-strand breaks by γ-H2AX immunofluorescence. Transcriptomic changes were profiled by RNA-seq with differential expression and GO enrichment analyses. Autophagy and endoplasmic reticulum (ER) stress markers were assessed by immunofluorescent staining. Key differentially expressed genes were validated by qRT-PCR. CuPT suppressed JEG-3 proliferation in a time- and concentration-dependent manner and reduced migratory and invasive capacities versus controls. Under 80 nM for 72 h, cell-cycle phase distribution showed no significant changes, but late apoptosis increased and intracellular ROS levels rose, indicating oxidative stress. γ-H2AX foci were elevated, consistent with DNA damage. RNA-seq revealed 648 upregulated and 451 downregulated genes, with enrichment of ER-stress, unfolded-protein response, intrinsic apoptotic signaling, and autophagy pathways among upregulated genes, and suppression of oxygen-response, glycolytic/energy metabolism, sterol biosynthesis, and cell-adhesion/mitotic regulation among downregulated genes. Concurrently, CuPT activated pronounced autophagosome formation and an ER stress response. CuPT treatment downregulated CYTB, ND6, and Egln3 expressions and upregulated NUPR1 and SQSTM1 expressions. CuPT impairs trophoblast-like JEG-3 cell proliferation, migration, and invasion, accompanied by increased ROS and DNA damage. Integrated RNA-seq and staining data indicate activation of ER stress and autophagy.
Isoflurane is a widely used inhalational anesthetic. Occupational exposure to waste anesthetic gases (WAGs) raises concerns about DNA damage. Bone marrow, highly susceptible due to its proliferative activity, has been scarcely investigated in rodents after exposure to WAGs. Therefore, the present study evaluated the impact of this exposure on micronucleus (MN) frequency and cytotoxicity, under conditions that mimic work environments with WAG to which thousands of professionals worldwide are occupationally exposed. Swiss mice were randomized into control, exposed, and recovery groups. Animals were subjected to daily 5 h exposures to 50 ppm isoflurane for 30 days. Bone marrow samples were analyzed using the MN assay. Isoflurane-exposed mice showed a significant increase in micronucleated polychromatic erythrocytes compared to controls (p < 0.0001). The recovery group exhibited a partial reduction after 20 days without exposure, though values remained above the control. Simulated occupational isoflurane exposure induces chromosomal instability in bone marrow cells, even at recommended safety levels.
Alternative DNA structure-forming (i.e., non-B) sequences such as H-DNA-forming sequences are enriched at chromosomal translocation hotspots in human cancer genomes, underscoring their role in genomic instability. H-DNA is particularly susceptible to DNA damage by reactive oxygen species (ROS), a common byproduct from both endogenous metabolism and environmental contaminants, thereby exacerbating its mutagenic potential. Oxidative lesions within B-DNA are efficiently processed by base excision repair (BER), whereas H-DNA is processed in a mutagenic fashion by nucleotide excision repair (NER). Thus, we speculate that the repair of oxidative lesions within H-DNA will promote aberrant BER and NER processing, ultimately enhancing mutagenesis. Here, we examine the processing of oxidative damage within H-DNA by measuring the changes in mutation frequencies and spectra, as well as the association with key NER and BER proteins in human cells in the presence or absence of specific DNA repair proteins. Our results demonstrate that oxidatively damaged H-DNA serves as a substrate for both BER and NER and reveals an interplay between BER and NER proteins, which influences mutation outcomes. This novel framework establishes a link between oxidative stress, DNA repair, and H-DNA-associated mutagenesis, providing insight into how environmentally relevant DNA damage can drive sequence-specific genomic instability at cancer-associated hotspots.
The Benchmark Dose (BMD) approach is commonly used to determine Point-of-Departure (PoD) values for risk assessment and regulatory decision-making; however, choosing a suitable Benchmark Response (BMR) for continuous endpoints is a challenge. Earlier work established a BMR of 50% for selected in vivo mutagenicity endpoints (i.e., Transgenic Rodent and Pig-a). Error-corrected sequencing (ECS) technologies, such as Duplex Sequencing (DupSeq), Hawk-Seq, PECC-Seq, and PacBio HiFi, have emerged as powerful tools for mutagenicity assessment. This study applied and compared two approaches for defining BMR values for ECS technologies: the Effect Size (ES) theory of Slob (2017), and the one standard deviation approach of Zeller et al. (2017). A dose-response database of ECS studies was compiled to determine technology-specific within-group variance values (var) for BMR determination. Experimental factor influences on var, including species, rodent strain, administration route, application time, tissue type, tissue sampling time, and DNA fragmentation method, were examined; no significant influences were detected. The absence of covariate effects justified using typical, technology-specific var values for BMR determinations. Using these values, technology-specific BMRs were calculated as 27.7% for DupSeq, 16.6% for Hawk-Seq, and 23.3% for PECC-Seq. BMRs derived from negative control values were 22.6 to 28.8% for DupSeq, 5.6 to 13.8% for Hawk-Seq, 28.7 to 31.5% for PECC-Seq, and 9.5 to 22.8% for PacBio HiFi. These findings support adoption of a 30% BMR for in vivo ECS mutagenicity assessment technologies, providing a robust and consistent foundation for future dose-response modeling and human health risk assessment.
The scientific concept of One Environmental Health is a research strategy focused on the study of toxicants, aiming to incorporate human, wildlife, and ecosystem health to establish a more comprehensive understanding of health. A One Environmental Health approach, studying the responses of American alligators (Alligator mississippiensis) that are considered apex sentinel organisms to environmental toxicants, is crucial. Due to their long-lived lifestyle on land and water, including a wide range of prey items in their diet and their ability to bioaccumulate metals, alligators serve as effective indicators of heavy metal pollution, which poses risks to aquatic ecosystems and human health. In this study, we examined DNA damage in American alligators from three locations in Florida using comet assay. We found alligators in Merritt Island National Wildlife Refuge had significantly higher DNA damage levels compared to those in Lake Apopka and Lake Woodruff. This trend was consistent across both sexes, with no observed sex differences. Similarly, DNA damage levels were significantly higher in both adult and juvenile alligators from Merritt Island compared to the other locations. Notably, juvenile alligators exhibited higher DNA damage than adults, with animals from Merritt Island exhibiting particularly elevated levels.
Alizarin is an anthraquinone red dye from natural or synthetic sources, widely used in textiles. Effluents of this activity can contain residual dyes, which may contaminate the aquatic environment. Studies report alizarin's aquatic toxicity, mutagenic, and carcinogenic effects. This study aimed to complement the aquatic toxicity evaluation and confirm its ability to cause genotoxicity in alternative models. Acute toxicity was performed with crustaceans, mussels, and fish embryos, while chronic toxicity was assessed in algae. Light effects on toxicity were evaluated using Daphnia similis. Histopathological effects on the gonads of Mytilus galloprovincialis and somatic mutations and sperm genotoxicity in Parhyale hawaiensis were investigated. Mutagenicity was confirmed using a miniaturized Ames test. The effect concentration 50% (EC50) for D. similis was 90.3, 105, and 68.6 μg L-1 for photoperiod (16 h light:8 h dark), light and dark, respectively. For Danio rerio embryos, the lethal concentration 50% (LC50) was 45.8 μg L-1, and an EC10 of 20.8 μg L-1 was calculated for sublethal effects. In vivo exposures caused alterations in the digestive gland and gonads of M. galloprovincialis, even in a short-term exposure, and increased the frequency of micronuclei and DNA damage in hemocytes and spermatozoids, respectively, of P. hawaiensis. It was mutagenic in the miniaturized Ames test using strain TA1537 (10% and 30% S9). Alizarin can be classified as a Category 1 acute aquatic toxicity according to the globally harmonized system (GHS). Due to adverse histopathological and DNA effects on reproductive systems in model organisms, it is considered a potential germ cell mutagen.
