Genotoxic Response Research Articles

Aliens Among Us: Sensitivity of the Invasive Alien Fish Black Bullhead Ameiurus melas as a Bioindicator of Pollution and Its Safety for Human Consumption

This study aims to evaluate the black bullhead Ameiurus melas, an invasive alien fish (IAF) in Serbia, as a bioindicator organism and assess the safety of natural and aquaculture specimens for human consumption. A set of biomarkers was analysed to assess the bioindicator potential at a site exposed to agricultural activities. The genotoxic response was determined by an alkaline comet assay and micronucleus assay in fish erythrocytes, and the metal pollution index (MPI) was calculated to assess the toxic element burden on fish. Water quality was evaluated using physicochemical parameters and faecal indicator bacteria, while sediment was analysed for the presence of pesticides. The concentration of metals and metalloids in fish muscle was monitored to assess the safety for human consumption, and the corresponding indices (MAC, THQ, HI) were calculated. All biomarker responses were linked by the integrated biomarker response (IBR). Water analyses indicated the absence of communal wastewater, while sediment analysis revealed the presence of paclobutrazol, bifenthrin, and cyfluthrin. The IBR showed that June and September had the highest stress indices, coinciding with peak pesticide use and precipitation. All indices confirmed the safety of black bullhead for human consumption. This study highlighted the uses of nature-based solutions to the problem of IAF.

Nuclear abnormalities and DNA damage indicate different genotoxic stress responses of marsh frogs (Pelophylax ridibundus, Pallas 1771) to industrial and agricultural water pollution in South Bulgaria.

Amphibians are continuously exposed to pollutants and anthropogenic stressors in their natural habitats, representing a significant challenge to their survival. This study aimed to quantify the extent of DNA damage caused by chronic industrial and agrochemical surface water pollution in wild populations of the marsh frog (Pelophylax ridibundus). The observed genotoxic effects on the marsh frog DNA, manifesting as abnormalities in erythrocyte nuclei, micronuclei, and DNA strand breaks, demonstrate a clear cause-and-effect relationship with surface water parameters, heavy metals, metalloids, and pesticides. The most prevalent nuclear abnormalities observed were notched and blebbed nuclei and nuclear buds, indicative of chromosomal instability. The significant correlation between cadmium, lead, and copper contamination and the increased frequency of DNA breakage in the marsh frogs from the industrial site indicates that heavy metal contamination has a higher genotoxic potential than pesticide contamination. These findings underscore the vulnerability of amphibians inhabiting heavy metal-contaminated wetlands to genotoxic stress due to their lower tolerance to environmental genotoxins. Therefore, using in situ assays to detect erythrocyte nuclear abnormalities and DNA damage in P. ridibundus could serve as a reliable indicator of environmental quality and provide early detection of anthropogenic pollution.

Sole and co-exposure toxicity of commercial formulations ethoprophos and bispyribac-sodium to Oreochromis niloticus: Assessment of oxidative stress, genotoxicity, and gill ultrastructure.

Aquatic organisms are simultaneously exposed to multiple hazardous chemicals that can be released into water bodies. The current study aimed to evaluate the effect of sublethal concentration (1/50 96h-LC50) of two formulated pesticides: ethoprophos, bispyribac-sodium, and their combination for 1, 2, 3, and 4weeks on oxidative stress, genotoxic response, and gill morphology in Nile tilapia. This study is the first to demonstrate the toxic effects of ethoprophos and bispyribac-sodium mixture on the commercial important species, Oreochromis niloticus. The results showed that the 96h-LC50 values of ethoprophos and bispyribac-sodium were 4.8 and 0.064mg/L, respectively. Additionally, exposure to individual or combined pesticides induced a significant increase in the level of malondialdehyde (MDA), glutathione S-transferase (GST), and 8-hydroxy-2-deoxyguanosine (8-OHdG), as well as a notable decline in reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels at all time of exposure. Furthermore, there were alterations in ultrastructure of the gill samples, including erosive lesions on the primary and secondary lamellae, fusion of microridges, and excessive mucus secretions on the epithelium. The data clearly demonstrate that the negative effects of the tested compounds are time-dependent and are more severe in combination than in a single compound. Collectively, our results indicated that the interaction of ethoprophos and bispyribac-sodium might be largely synergistic and provide new insights into the molecular mechanisms of fish confronting these substances.

The Toxic Effects of Petroleum Diesel, Biodiesel, and Renewable Diesel Exhaust Particles on Human Alveolar Epithelial Cells.

The use of alternative diesel fuels has increased due to the demand for renewable energy sources. There is limited knowledge regarding the potential health effects caused by exhaust emissions from biodiesel- and renewable diesel-fueled engines. This study investigates the toxic effects of particulate matter (PM) emissions from a diesel engine powered by conventional petroleum diesel fuel (SD10) and two biodiesel and renewable diesel fuels in vitro. The fuels used were rapeseed methyl ester (RME), soy methyl ester (SME), and Hydrogenated Vegetable Oil (HVO), either pure or as 50% blends with SD10. Additionally, a 5% RME blend was also used. The highest concentration of polycyclic aromatic hydrocarbon emissions and elemental carbon (EC) was found in conventional diesel and the 5% RME blend. HVO PM samples also exhibited a high amount of EC. A dose-dependent genotoxic response was detected with PM from SD10, pure SME, and RME as well as their blends. Reactive oxygen species levels were several times higher in cells exposed to PM from SD10, pure HVO, and especially the 5% RME blend. Apoptotic cell death was observed in cells exposed to PM from SD10, 5% RME blend, the 50% SME blend, and HVO samples. In conclusion, all diesel PM samples, including biodiesel and renewable diesel fuels, exhibited toxicity.

Gene expression in soft-shell clam (Mya arenaria) transmissible cancer reveals survival mechanisms during host infection and seawater transfer.

Transmissible cancers are unique instances in which cancer cells escape their original host and spread through a population as a clonal lineage, documented in Tasmanian Devils, dogs, and ten bivalve species. For a cancer to repeatedly transmit to new hosts, these lineages must evade strong barriers to transmission, notably the metastasis-like physical transfer to a new host body and rejection by that host's immune system. We quantified gene expression in a transmissible cancer lineage that has spread through the soft-shell clam (Mya arenaria) population to investigate potential drivers of its success as a transmissible cancer lineage, observing extensive differential expression of genes and gene pathways. We observed upregulation of genes involved with genotoxic stress response, ribosome biogenesis and RNA processing, and downregulation of genes involved in tumor suppression, cell adhesion, and immune response. We also observe evidence that widespread genome instability affects the cancer transcriptome via gene fusions, copy number variation, and transposable element insertions. Finally, we incubated cancer cells in seawater, the presumed host-to-host transmission vector, and observed conserved responses to halt metabolism, avoid apoptosis and survive the low-nutrient environment. Interestingly, many of these responses are also present in healthy clam cells, suggesting that bivalve hemocytes may have inherent seawater survival responses that may partially explain why transmissible cancers are so common in bivalves. Overall, this study reveals multiple mechanisms this lineage may have evolved to successfully spread through the soft-shell clam population as a contagious cancer, utilizing pathways known to be conserved in human cancers as well as pathways unique to long-lived transmissible cancers.

GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida.

Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse Pseudomonas putida that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.

Genotoxic stress impacts pre-mRNA 3'-end processing.

Genotoxic stress, arising from various environmental sources and endogenous cellular processes, pose a constant threat to genomic stability. Cells have evolved intricate mechanisms to detect and repair DNA damage, orchestrating a robust genotoxic stress response to safeguard the integrity of the genome. Recent research has shed light on the crucial role of co- and post-transcriptional regulatory mechanisms in modulating the cellular response to genotoxic stress. Here we highlight recent advances illustrating the intricate interplay between pre-mRNA processing, with a focus on 3'-end processing, and genotoxic stress response.

Arabidopsis Histone Variant H2A.X Functions in the DNA Damage-Coupling Abscisic Acid Signaling Pathway.

Environmental variations initiate chromatin modifications, leading to the exchange of histone subunits or the repositioning of nucleosomes. The phosphorylated histone variant H2A.X (γH2A.X) is recognized for the formation of foci that serve as established markers of DNA double-strand breaks (DSBs). Nevertheless, the precise roles of H2A.X in the cellular response to genotoxic stress and the impact of the plant hormone abscisic acid (ABA) remain incompletely understood. In this investigation, we implemented CRISPR/Cas9 technology to produce loss-of-function mutants of AtHTA3 and AtHTA5 in Arabidopsis. The phenotypes of the athta3 and athta5 single mutants were nearly identical to those of the wild-type Col-0. Nevertheless, the athta3 athta5 double mutants exhibited aberrant embryonic development, increased sensitivity to DNA damage, and higher sensitivity to ABA. The RT-qPCR analysis indicates that AtHTA3 and AtHTA5 negatively regulate the expression of AtABI3, a fundamental regulator in the ABA signaling pathway. Subsequent investigation demonstrated that AtABI3 participates in the genotoxic stress response by influencing the expression of DNA damage response genes, such as AtBRCA1, AtRAD51, and AtWEE1. Our research offers new insights into the role of H2A.X in the genotoxic and ABA responses of Arabidopsis.

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 (Polɩ) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of Polɩ and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding Polɩ 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 Polɩ 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.

ADAR-Mediated A>I(G) RNA Editing in the Genotoxic Drug Response of Breast Cancer.

Epitranscriptomics is a field that delves into post-transcriptional changes. Among these modifications, the conversion of adenosine to inosine, traduced as guanosine (A>I(G)), is one of the known RNA-editing mechanisms, catalyzed by ADARs. This type of RNA editing is the most common type of editing in mammals and contributes to biological diversity. Disruption in the A>I(G) RNA-editing balance has been linked to diseases, including several types of cancer. Drug resistance in patients with cancer represents a significant public health concern, contributing to increased mortality rates resulting from therapy non-responsiveness and disease progression, representing the greatest challenge for researchers in this field. The A>I(G) RNA editing is involved in several mechanisms over the immunotherapy and genotoxic drug response and drug resistance. This review investigates the relationship between ADAR1 and specific A>I(G) RNA-edited sites, focusing particularly on breast cancer, and the impact of these sites on DNA damage repair and the immune response over anti-cancer therapy. We address the underlying mechanisms, bioinformatics, and in vitro strategies for the identification and validation of A>I(G) RNA-edited sites. We gathered databases related to A>I(G) RNA editing and cancer and discussed the potential clinical and research implications of understanding A>I(G) RNA-editing patterns. Understanding the intricate role of ADAR1-mediated A>I(G) RNA editing in breast cancer holds significant promise for the development of personalized treatment approaches tailored to individual patients' A>I(G) RNA-editing profiles.

Chemical Composition of PM2.5-0.3 and PM0.3 Collected in Southern Lebanon and Assessment of Their Toxicity in BEAS-2B Cells

Fine particles (PM2.5) have generally been reported as the major contributor to the adverse health effects of air pollution. Lebanon is characterized by a high density of transport, the production of electricity by generators, and a problem of uncontrolled incineration of household waste. For the purpose of this paper, the physico-chemical properties of fine (PM2.5-0.3) and quasi-ultrafine (PM0.3) particulate matter sampled in Southern Lebanon, were studied. Then, an evaluation and comparison of the toxicity of the different extracted fractions from PM (i.e., native PM2.5-0.3 vs. organic extractable matter fraction (OEM2.5-0.3), and non-extractable matter fraction (NEM2.5-0.3)) was performed. Also, an examination of the toxicity of PM0.3 was conducted indirectly through the evaluation of the OEM0.3 harmfulness. The physico-chemical analysis showed that PM0.3 was much more concentrated than PM2.5-0.3 in organic compounds such as polycyclic aromatic hydrocarbons (PAHs) (28-fold) and their nitrated (N-PAHs, 14-fold) and oxygenated (O-PAHs, 10-fold) derivatives. Normal human bronchial epithelial cells (BEAS-2B) were exposed to PM2.5-0.3, its derived fractions (i.e., OEM2.5-0.3 and NEM2.5-0.3), and OEM0.3 before evaluating the global cytotoxicity, metabolic activation of organic compounds, genotoxicity, and inflammatory response. Different responses were observed depending on the considered fraction of particles. The global cytotoxicity showed a pronounced response related to ATP and LDH activities after exposure to the quasi-ultrafine organic extractable matter fraction (OEM0.3). There was no significant induction of the AhR cell-signaling pathway by NEM2.5-0.3. Despite the apparent difference in the kinetics of induction of the toxicological endpoints under study, OEM0.3 provoked a higher overall cytotoxicity and genotoxicity than OEM2.5-0.3 and total PM2.5-0.3. Taken together, these results clearly showed that the finest particles are more damaging to BEAS-2B cells than PM2.5-0.3 because they are richer in organic compounds, thereby inducing more remarkable toxic effects.

Oxidative Stress Responses in Obese Individuals Undergoing Bariatric Surgery: Impact on Carcinogenesis.

Obesity is a big public health problem that claims several thousand lives every year. Bariatric surgery has arisen as a suitable procedure for treating obesity, particularly morbid obesity. Oxidative stress, genotoxicity, apoptosis, and inflammatory responses are recognized as the most important occurrences in carcinogenesis, as they actively contribute to the multistep process. This study aimed to briefly review the connection between oxidative stress, genotoxicity, apoptosis, and inflammation in obese patients undergoing bariatric surgery, focusing on its impact on carcinogenesis. Regarding oxidative stress, bariatric surgery may inhibit the synthesis of reactive oxygen species. Moreover, a significant reduction in the inflammatory status after weight loss surgery was not observed. Bariatric surgery prevents apoptosis in several tissues, but the maintenance of low body weight for long periods is mandatory for mitigating DNA damage. In conclusion, the association between bariatric surgery and cancer risk is still premature. However, further studies are yet needed to elucidate the real association between bariatric surgery and a reduced risk of cancer.

Exploring the safety of cannabidiol (CBD): A comprehensive in vitro evaluation of the genotoxic and mutagenic potential of a CBD isolate and extract from Cannabis sativa L

Cannabidiol (CBD), a naturally occurring cyclic terpenoid found in Cannabis sativa L., is renowned for its diverse pharmacological benefits. Marketed as a remedy for various health issues, CBD products are utilized by patients as a supplementary therapy or post-treatment failure, as well as by healthy individuals seeking promised advantages. Despite its widespread use, information regarding potential adverse effects, especially genotoxic properties, is limited. The present study is focused on the mutagenic and genotoxic activity of a CBD isolate (99.4 % CBD content) and CBD-rich Cannabis sativa L extract (63.6 % CBD content) in vitro. Both CBD samples were non-mutagenic, as determined by the AMES test (OECD 471) but exhibited cytotoxicity for HepG2 cells (∼IC50(4 h) 26 µg/ml, ∼IC50(24 h) 6–8 µg/ml, MTT assay). Noncytotoxic concentrations induced upregulation of genes encoding metabolic enzymes involved in CBD metabolism, and CBD oxidative as well as glucuronide metabolites were found in cell culture media, demonstrating the ability of HepG2 cells to metabolize CBD. In this study, the CBD samples were found non-genotoxic. No DNA damage was observed with the comet assay, and no influence on genomic instability was observed with the cytokinesis block micronucleus and the γH2AX and p-H3 assays. Furthermore, no changes in the expression of genes involved in genotoxic stress response were detected in the toxicogenomic analysis, after 4 and 24 h of exposure. Our comprehensive study contributes valuable insights into CBD's safety profile, paving the way for further exploration of CBD's therapeutic applications and potential adverse effects.

Acute Toxicity and Genotoxic Responses in African Mud Catfish, Clarias gariepinus, Exposed to Biocides and Aqueous Ammonia

Acute Toxicity and Genotoxic Responses in African Mud Catfish, Clarias gariepinus, Exposed to Biocides and Aqueous Ammonia

Evaluation of cytotoxicity, acute toxicity, genotoxicity and antioxidant and antigenotoxicity activities of the sarcotesta fraction of punica granatum L. rich in lectin (PgTel)

ABSTRACT Punica granatum, popularly known as pomegranate, is a fruit tree with wide worldwide distribution, containing numerous phytochemicals of great medicinal value. The aim of the present study was to determine the phytochemical profile and antioxidant potential of a protein fraction (PF) derived from P. granatum sarcotesta which is rich in lectin. In addition, the acute oral toxicity, genotoxicity and antigenotoxicity of this protein fraction (PF) from P. granatum sarcotesta was measured. The phytochemical profile of PF was determined using HPLC. The in vitro antioxidant effect was assessed using the methods of total antioxidant capacity (TAC) and DPPH and ABTS+ radical scavenging. Acute oral toxicity was determined in female Swiss mice administered a single dose of 2000 mg/kg. This PF was examined for genotoxicity and antigenotoxicity at doses of 500, 1000 and 2000 mg/kg, utilizing mouse peripheral blood cells. Phytochemical characterization detected a high content of ellagic acid and antioxidant capacity similar to that of ascorbic acid (positive control). PF was not toxic (LD50 >2000 mg/kg) and did not exert a genotoxic effect in mice. PF protected the DNA of peripheral blood cells against damage induced by cyclophosphamide. In conclusion, this PF fraction exhibited significant antioxidant activity without initiating toxic or genotoxic responses in mice.

Toxicity assessment of animal manure composts containing environmental microplastics by using earthworms Eisenia andrei

Nowadays, animal manure composting constitutes a sustainable alternative for farmers to enhance the level of nutrients within soils and achieve a good productivity. However, pollutants may be present in manures. This study focuses on the detection of environmental microplastics (EMPs) into composts, as well as on the assessment of their potential toxicity on the earthworm Eisenia andrei. To these aims, animals were exposed to two types of compost, namely bovine (cow) and ovine (sheep) manure, besides to their mixture, for 7 and 14 days. The presence and characterization of EMPs was evaluated in all the tested composts, as well as in tissues of the exposed earthworms. The impact of the tested composts was assessed by a multi-biomarker approach including cytotoxic (lysosomal membrane stability, LMS), genotoxic (micronuclei frequency, MNi), biochemical (activity of catalase, CAT, and glutathione-S-transferase, GST; content of malondialdehyde, MDA), and neurotoxic (activity of acetylcholinesterase, AChE) responses in earthworms. Results indicated the presence of high levels of EMPs in all the tested composts, especially in the sheep manure (2273.14 ± 200.89 items/kg) in comparison to the cow manure (1628.82 ± 175.23 items/kg), with the size <1.22 μm as the most abundant EMPs. A time-dependent decrease in LMS and AChE was noted in exposed earthworms, as well as a concomitant increase in DNA damages (MNi) after 7 and 14 days of exposure. Also, a severe oxidative stress was recorded in animals treated with the different types of compost through an increase in CAT and GST activities, and LPO levels, especially after 14 days of exposure. Therefore, it is necessary to carefully consider these findings for agricultural good practices in terms of plastic mitigation in compost usage, in order to prevent any risk for environment health.

Occupational second-hand smoke exposure: A comparative shotgun proteomics study on nasal epithelia from healthy restaurant workers

Non-smokers exposed to second-hand smoke (SHS) present risk of developing tobacco smoke-associated pathologies. To investigate the airway molecular response to SHS exposure that could be used in health risk assessment, comparative shotgun proteomics was performed on nasal epithelium from a group of healthy restaurant workers, non-smokers (never and former) exposed and not exposed to SHS in the workplace. HIF1α-glycolytic targets (GAPDH, TPI) and proteins related to xenobiotic metabolism, cell proliferation and differentiation leading to cancer (ADH1C, TUBB4B, EEF2) showed significant modulation in non-smokers exposed. In never smokers exposed, enrichment of glutathione metabolism pathway and EEF2-regulating protein synthesis in genotoxic response were increased, while in former smokers exposed, proteins (LYZ, ATP1A1, SERPINB3) associated with tissue damage/regeneration, apoptosis inhibition and inflammation that may lead to asthma, COPD or cancer, were upregulated. The identified proteins are potential response and susceptibility/risk biomarkers for SHS exposure.

Genotoxic Response and Histological Alterations in Rat Lungs Exposed to Gasoline Generator Exhaust

The epileptic power supply in Nigeria has caused a surge in the production of energy from other sources, including gasoline generator sets. The air pollution caused by the exhaust from these engines has been implicated in a variety of metabolic disorders and diseases, including cancer. This study aims at understanding the genotoxic and histopathological effects of gasoline generator exhaust exposure on the DNA and lungs of adult male wistar rats. Forty- eight (48) adult wistar rats were divided into four categories, each with 12 rats. The control rats were not exposed to gasoline generator exhaust while the test groups were exposed at 2, 4, and 8-hour intervals for a duration of 4, 8, and, 12 weeks respectively. Animals were euthanized using cervical dislocation and blood samples were obtained for genotoxic analysis via cardiac puncture while the lungs were preserved for 72 hours in 10% neutral buffered formalin and further processed for histological studies. The Olive Tail Moment (OTM) of the exposed rats showed significant variation across the exposure time points when compared to the unexposed control, indicating that exposure to gasoline generator exhaust negatively impacts the DNA. Additionally, histological investigations also indicated some cytopathic characteristics, such as peribronchiolar and perivascular infiltration by inflammatory cells, congestion, and thickening of blood vessels. The findings of this study showed that exposure to gasoline generator exhaust induces genotoxic damage with the severity of the damage occurring in a time/exposure-dependent manner. Keywords: Air pollution; Nigeria; Gasoline; Generator; Genotoxicity.

Liver-on-chip model and application in predictive genotoxicity and mutagenicity of drugs

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.

A scenario of unhealthy life cycle: The role of circadian rhythms in health.

Circadian rhythms are oscillations in physiology and behavior caused by the circadian regulator. Cryptochromes, Periods, and Bmal1 are circadian clock genes that have been linked to aging and cancer. Human pathologies alter circadian clock gene expression, and transgenic rats with clock gene defects progress to cancer and age prematurely. In the growth of age-linked pathologies and carcinogenesis, cell proliferation and genome integrity play critical roles. The relationship concerning the cell cycle regulation and circadian clock is discussed in this article. The circadian clock controls the behavior and countenance of many main cell cycle and cell cycle check-point proteins, and cell cycle-associated proteins, in turn, control the activity and expression of circadian clock proteins. The circadian clock can be reset by DNA disruption, providing a molecular mechanism for mutual control amid the cell cycle and the clock. This circadian clock-dependent regulation of cell proliferation, composed with other circadian clock-dependent physiological functions including metabolism control, genotoxic and oxidative stress response, and DNA repair, unlocks new avenues for studying the processes of aging and carcinogenesis.