Key Challenges for In Vitro Testing of Tobacco Products for Regulatory Applications: Recommendations for the In Vitro Mouse Lymphoma Assay.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across traditional tobacco and various tobacco and nicotine next-generation products (NGPs), including Heated Tobacco Products (HTPs) and Electronic Nicotine Delivery Systems (ENDS). This report was developed by a working group composed of attendees of the seventh IIVS workshop, 'Approaches and recommendations for conducting the mouse lymphoma gene mutation assay (MLA) and introduction to in vitro disease models', which was held virtually on 21-23 June 2022. This publication provides a background overview of the MLA, and includes the description of assay conduct and data interpretation, key challenges and recommended best practices for evaluating tobacco and nicotine products, with a focus on the evaluation of NGPs, and a summary of how the assay has been used to evaluate and compare tobacco and nicotine products.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Collecting e-cigarette aerosols for in vitro applications: A survey of the biomedical literature and opportunities to increase the value of submerged cell culture-based assessments.

Electronic nicotine delivery systems (ENDS) are being developed as potentially reduced-risk alternatives to the continued use of combustible tobacco products. Because of the widespread uptake of ENDS-in particular, e-cigarettes-the biological effects, including the toxic potential, of their aerosols are under investigation. Preclinically, collection of such aerosols is a prerequisite for testing in submerged cell culture-based in vitro assays; however, despite the growth in this research area, there is no apparent standardized collection method for this application. To this end, through an Institute for in vitro Sciences, Inc. workshop initiative, we surveyed the biomedical literature catalogued in PubMed® to map the types of methods hitherto used and reported publicly. From the 47 relevant publications retrieved, we identified seven distinct collection methods. Bubble-through (with aqueous solvents) and Cambridge filter pad (CFP) (with polar solvents) collection were the most frequently cited methods (57% and 18%, respectively), while the five others (CFP + bubble-through; condensation; cotton filters; settle-upon; settle-upon + dry) were cited less often (2-10%). Critically, the collected aerosol fractions were generally found to be only minimally characterized chemically, if at all. Furthermore, there was large heterogeneity among other experimental parameters (e.g., vaping regimen). Consequently, we recommend that more comprehensive research be conducted to identify the method(s) that produce the fraction(s) most representative of the native aerosol. We also endorse standardization of the aerosol generation process. These should be regarded as opportunities for increasing the value of in vitro assessments in relation to predicting effects on human health.

Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro.

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

Lysosomotropic-related limitations of the BALB/c 3T3 cell-based neutral red uptake assay and an alternative testing approach for assessing e-liquid cytotoxicity.

Cytotoxicity assays are used to quantify the cytotoxic potential of chemicals. The neutral red uptake (NRU) assay is one of these assays and is routinely used in the pharmaceutical, cosmetic, and tobacco industries. In the context of e-cigarette development, an NRU assay-based screen was implemented to evaluate the cytotoxic potential of e-liquids. E-liquids induced a biphasic response in the BALB/c 3T3-based assay. The NRU initially increased in a concentration-dependent manner before decreasing following treatment with higher concentrations until NRU was abolished. Experiments were performed to characterize the mechanism underlying this biphasic signal. Nicotine alone was found to induce the same biphasic effects, while inducing concentration-dependent decreases in relative cell counts (RCC). Imaging and flow cytometry data revealed that the increases in NRU likely resulted from nicotine-induced vacuolization via a lysosomotropic mechanism. In support of this, two lysosomotropic agents, chloroquine and lapatinib, induced similar profiles. Nicotine's effects were also translatable, as brain-, lung-, bone marrow-, and smooth muscle-derived mammalian cells responded with the biphasic NRU signal. However, like RCC, three other cytotoxicity endpoints, resazurin, adenosine triphosphate, and water soluble tetrazolium salt (WST)-8, were not subject to these effects. The WST-8 assay is proposed as an alternative to screen the cytotoxic potential of e-liquids.

Mode-of-action analysis of the effects induced by nicotine in the in vitro micronucleus assay.

Nicotine's genotoxic potential has been extensively studied in vitro. While the results of mammalian cell-based studies have inferred that it can potentially damage chromosomes, in general and with few exceptions, adverse DNA effects have been observed primarily at supraphysiological concentrations in nonregulatory assays that provide little information on its mode-of-action (MoA). In this study, a modern-day regulatory genotoxicity assessment was conducted using a flow cytometry-based in vitro micronucleus (MN) assay, Good Laboratory Practice study conditions, Chinese hamster ovary cells of known provenance, and acceptance/evaluation criteria from the current OECD Test Guideline 487. Nicotine concentrations up to 3.95 mM had no effect on background levels of DNA damage; however, concentrations above the point-of-departure range of 3.94-4.54 mM induced increases in MN and hypodiploid nuclei, indicating a possible aneugenicity hazard. Follow-up experiments designed to elucidate nicotine's MoA revealed cellular vacuolization, accompanying distortions in microtubules, inhibition of tubulin polymerization, centromere-positive DNA, and multinucleate cells at MN-inducing concentrations. Vacuoles likely originated from acidic cellular compartments (e.g., lysosomes). Remarkably, genotoxicity was suppressed by chemicals that raised the luminal pH of these organelles. Other endpoints (e.g., changes in phosphorylated histones) measured in the study cast doubt on the biological relevance of this apparent genotoxicity. In addition, three major nicotine metabolites, including cotinine, had no MN effects but nornicotine induced a nicotine-like profile. It is possible that nicotine's lysosomotropic properties drive the genotoxicity observed in vitro; however, the potency and mechanistic insights revealed here indicate that it is likely of minimal physiological relevance for nicotine consumers. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

The Effects of Sleep Extension on Sleep, Performance, Immunity and Physical Stress in Rugby Players.

(1) Background: The purpose of the present study was to examine the efficacy of sleep extension in professional rugby players. The aims were to: (i) characterise sleep quantity in elite rugby players and determine changes in immune function and stress hormone secretion during a pre-season training programme; (ii) evaluate the efficacy of a sleep extension intervention in improving sleep, markers of physical stress, immune function and performance. (2) Methods: Twenty five highly trained athletes from a professional rugby team (age (mean ± SD) 25 ± 2.7 years; height 1.87 ± 0.07 m; weight 105 ± 12.1 kg) participated in a six week pre-post control-trial intervention study. Variables of sleep, immune function, sympathetic nervous activity, physiological stress and reaction times were measured. (3) Results: Sleep extension resulted in a moderate improvement in sleep quality scores ([mean; ± 90% confidence limits] −24.8%; ± 54.1%) and small to moderate increases in total sleep time (6.3%; ± 6.3%) and time in bed (7.3%; ± 3.6%). In addition, a small decrease in cortisol (−18.7%; ± 26.4%) and mean reaction times (−4.3%; ± 3.1%) was observed following the intervention, compared to the control. (4) Conclusions: Professional rugby players are at risk of poor sleep during pre-season training, with concomitant rises in physical stress. Implementing a sleep extension programme among professional athletes is recommended to improve sleep, with beneficial changes in stress hormone expression and reaction time performance.

Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol.

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

Prevalence of poor sleep quality, sleepiness and obstructive sleep apnoea risk factors in athletes.

PURPOSE: Despite the perceived importance of sleep for athletes, little is known regarding athlete sleep quality, their prevalence of daytime sleepiness or risk factors for obstructive sleep apnoea (OSA) such as snoring and witnessed apnoeic episodes. The purpose of the present study was to characterise normative sleep quality among highly trained team sport athletes. METHODOLOGY: 175 elite or highly trained rugby sevens, rugby union and cricket athletes completed the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Score (ESS) and Quality of Life questionnaires and an OSA risk factor screen. RESULTS: On average, athletes reported 7.9 ± 1.3 h of sleep per night. The average PSQI score was 5.9 ± 2.6, and 50% of athletes were found to be poor sleepers (PSQI > 5). Daytime sleepiness was prevalent throughout the population (average global score of 8.5) and clinically significant (ESS score of ≥10) in 28% of athletes. OSA may be an important clinical consideration within athletic populations, as a considerable number of athletes (38%) defined themselves as snorers and 8% reported having a witnessed apnoeic episode. The relationship between self-rated sleep quality and actual PSQI score was strong (Pearson correlation of 0.4 ± 0.1, 90% confidence limits). CONCLUSION: These findings suggest that this cohort of team sport athletes suffer a preponderance of poor sleep quality, with associated high levels of daytime sleepiness. Athletes should receive education about how to improve sleep wake schedules, extend total sleep time and improve sleep quality.

The training effect of short term enhancement from complex pairing on horizontal and vertical countermovement and drop jump performance.

The purpose of this study was to investigate the chronic effect of a contrast training program designed to elicit an acute short term enhancement (STE) effect during training. A matched pairs training study design was implemented with a contrast (STE affected) and complex (control) training group completing a seven week training intervention. Twenty subjects participated. The contrast training group completed training based on a preloading protocol that had previously been shown to induce an acute STE effect within the subject population. The control group completed the same volume and type of training in a complex training format. Changes in squat 4RM strength and kinetic and kinematic performance in vertical and horizontal countermovement jump (CMJ) and drop jump (DJ) were measured via a Force Plate. Differences between the experimental and control group in change of mean strength (effect size 0.03 ± 0.33), vertical DJ (effect size: contact time -0.22 ± 0.52; peak force 0.20 ± 0.30; mean force 0.30 ± 0.74) and horizontal DJ (effect size: contact time -0.47 ± 0.73; peak force 0.03 ± 0.36; mean force 0.13 ± 0.56) were not meaningful. However, differences in mean change of vertical and horizontal CMJ measures of force (effect size range 0.40 - 0.46 ± 0.37 - 0.63), vertical CMJ peak velocity (effect size 0.84 ±0.66) and mean velocity (0.62 ± 0.88) were meaningful. These findings demonstrate that eliciting an acute STE response in dynamic training movements through contrast training can produce a chronic improvement in dynamic movements as a training effect.

Relationship between vertical and horizontal jump variables and muscular performance in athletes.

This study investigated the relationship between vertical and horizontal measures in bilateral and unilateral countermovement jump, drop jump and squat jump (SJ), and sprinting speed and muscle architecture of both the vastus lateralis and gastrocnemius. Subjects (n = 17) completed a 30-m sprint test, muscle stiffness test; ultrasound measures, and a jump testing session. Measures of horizontal peak and mean force, in both bilateral and unilateral jumps, tended to have greater relationships to sprint speeds (R = 0.132-0.576) than peak and mean force in the vertical plane (R = 0.008-0.504). Vertical velocity variables also showed some large and very large correlations to sprint speed (R = 0.062-0.635). Unilateral measures of velocity tended to have larger correlations to sprint performance than their bilateral counterparts across all jump types and peak and mean velocity in SJ showed large and very large correlations to sprint speed (bilateral R = 0.227-0.635; unilateral 0.393-0.574). Few large correlations were shown between muscle stiffness measures of muscle architecture and kinetic and kinematic variables in either vertical or horizontal jumps. The present findings suggest that sport scientists and strength and conditioning practitioners concerned with the prognostic value of kinetic variables to functional movements such as sprint speed should also use horizontal jumps in addition to vertical jumps in testing and training.

The relationship between physical fitness and game behaviours in rugby union players.

The physical preparation of team sport athletes should reflect the degree to which each component of fitness is relied upon in competition. The aim of the study was therefore to establish the relationship between fitness-test data and game behaviours known or thought to be important for successful play in rugby union matches. Fitness-test measures from 510 players were analysed with game statistics, from 296 games within the 2007 and 2008 calendar years. Sprint times over 10, 20 and 30 m had moderate to small negative correlations (r) with line breaks (~0.26), metres advanced (~0.22), tackle breaks (~0.16) and tries scored (~0.15). The average time of 12 repeated sprints and percentage body fat in the forwards, and repeated sprint fatigue in the backs had moderate to small correlations with a measure of activity rate on and around the ball (-0.38, -0.17 and -0.17, respectively). These low correlations are partly due to uniformly high physical fitness as a result of selection pressures at the elite level and leave room for the identification of other key predictors. Nonetheless, physical conditioning programmes should be adapted to reflect the importance of speed, repeated sprint ability and body composition in the performance of key game behaviours during competition.

Differences and changes in the physical characteristics of professional and amateur rugby union players.

Numerous studies have highlighted differences between playing levels and positions in rugby union; however, few studies have investigated longitudinal progressions of body composition and physical performance. Between-player differences and within-player changes in body composition, strength, power, speed, and repeated sprint ability, from 1,161 New Zealand rugby union players from 2004 to 2007, were estimated using a mixed modeling procedure. Props had the highest mass, percent body fat, strength, and slowest speed times compared with the other positions, whereas outside backs had the fastest speed time and lowest percent body fat. For most measures, there were small-to-moderate differences (range, 1.1-14%) between players selected and not selected for provincial teams and small-to-large differences (range, 1.8-15%) between provincial and Super Rugby (professional) players. The faster 20-m sprint times in international compared with Super Rugby players was small in magnitude for both the forwards (1.9%) and backs (2.2%). The average annual improvements were small to moderate for strength (range, 2.1-15%) and small for repeated sprint ability within the lower playing levels (~1.5%). Small increases occurred in lower body strength (~7.0%) as players moved from Super Rugby to provincial competition. Small decreases in sprint time (~1.6%) and small increases in strength (~6.3%) occurred as players moved from Super Rugby to midyear international competition. The differences between levels in performance provide level-specific characteristics from Super Rugby and below, but international players may be selected because of greater skill and experience. Changes in physical performance between competitions may be a result of reduced training loads because of regular high-intensity matches and greater travel involved in the Super Rugby competition.

Relationship of cellular topoisomerase IIα inhibition to cytotoxicity and published genotoxicity of fluoroquinolone antibiotics in V79 cells.

Fluoroquinolone (FQ) antibiotics are bacteriocidal through inhibition of the bacterial gyrase and at sufficient concentrations in vitro, they can inhibit the homologous eukaryotic topoisomerase (TOPO) II enzyme. FQ exert a variety of genotoxic effects in mammalian systems through mechanisms not yet established, but which are postulated to involve inhibition of TOPO II enzymes. To assess the relationship of inhibition of cell nuclear TOPO II to cytotoxicity and reported genotoxicity, two FQ, clinafloxacin (CLFX) and lomefloxacin (LOFX), having available genotoxicity data showing substantial differences with CLFX being more potent than LOFX, were selected for study. The relative inhibitory activities of these FQ on nuclear TOPO IIα in cultured Chinese hamster lung fibroblasts (V79 cells) over dose ranges and at equimolar concentrations were assessed by measuring nuclear stabilized cleavage complexes of TOPO IIα-DNA. Cytotoxicity was measured by relative cell counts. Both FQ inhibited V79 cell nuclear TOPO IIα. The lowest-observed-adverse-effect levels for TOPO IIα inhibition were 55 µM for CLFX, and 516 µM for LOFX. The no-observed-adverse-effect-levels were 41 µM for CLFX, and 258 µM for LOFX. At equimolar concentrations (175 µM), CLFX was more potent than LOFX. Likewise, CLFX was more cytotoxic than LOFX. Thus, the two FQ, inhibited TOPO IIα in intact V79 cells, differed in their potencies and exhibited no-observed-adverse-effect levels. These findings are in concordance with published genotoxicity data and observed cytotoxicity.

Effects of an off-season conditioning program on the physical characteristics of adolescent rugby union players.

The aims of the study were to determine if a supervised off-season conditioning program enhanced gains in physical characteristics compared with the same program performed in an unsupervised manner and to establish the persistence of the physical changes after a 6-month unsupervised competition period. Forty-four provincial representative adolescent rugby union players (age, mean ± SD, 15.3 ± 1.3 years) participated in a 15-week off-season conditioning program either under supervision from an experienced strength and conditioning coach or unsupervised. Measures of body composition, strength, vertical jump, speed, and anaerobic and aerobic running performance were taken, before, immediately after, and 6 months after the conditioning. Post conditioning program the supervised group had greater improvements in all strength measures than the unsupervised group, with small, moderate and large differences between the groups\x{2019} changes for chin-ups (9.1%; ± 11.6%), bench-press (16.9%; ± 11.7%) and box-squat (50.4%; ± 20.9%) estimated 1RM respectively. Both groups showed trivial increases in mass; however increases in fat free mass were small and trivial for supervised and unsupervised players respectively. Strength declined in the supervised group while the unsupervised group had small increases during the competition phase, resulting in only a small difference between the long-term changes in box-squat 1RM (15.9%; ± 13.2%). The supervised group had further small increases in fat free mass resulting in a small difference (2.4%; ± 2.7%) in the long-term changes. The postconditioning differences between the 2 groups may have been a result of increased adherence and the attainment of higher training loads during supervised training. The lack of differences in strength after the competition period indicates that supervision should be maintained to reduce substantial decrements in performance.

Evaluating the genotoxicity of topoisomerase-targeted antibiotics.

Antibiotics like fluoroquinolones (FQs) that target bacterial type II topoisomerases pose a potential genotoxic risk due to interactions with mammalian topoisomerase II (TOPO II) counterparts. Inhibition of TOPO II can lead to the generation of clastogenic DNA double-strand breaks (DSBs) that can in turn manifest in mutagenesis. Thus, methods that allow early identification of drugs that present the greatest hazard are warranted. A rapid, medium-throughput and predictive genotoxicity screen that can be applied to bacterial type II topoisomerase inhibitors is described herein. Maximal induction of the DSB biomarker serine139-phosphorylated histone H2AX (γH2AX) in L5178Y cells was quantified via flow cytometry and correlated with data derived from the mouse lymphoma screen (MLS), a default assay used to rank genotoxic potential. When applied to a class of novel bacterial type II topoisomerase inhibitors (NBTIs) in lead-optimisation, maximal γH2AX induction >1.4-fold (relative to controls) identified 22/27 NBTIs that induced >6-fold relative mutation frequency (MF) in MLS. Moreover, response signatures comprising of γH2AX induction and G(2)M cell cycle arrest elucidated using this approach suggested that these NBTIs, primarily of the H class, operated via a TOPO II poison-like mechanism of action (MoA) similar to FQs. NBTIs that induced ≤6-fold relative MF, which were mainly A class-derived, had less impact on γH2AX (≤1.4-fold) and also evoked G(1) arrest, indicating that their cytotoxic effects were likely mediated through a non-poison MoA. Concordance between assays was 86% (54/63) when 1.4- and 6-fold 'cut offs' were applied. These findings were corroborated through inspection of human TOPO IIα IC(50) data as NBTIs exhibiting equivalent inhibitory capacities had differing genotoxic potencies. Deployed in an early screening capacity, the γH2AX by flow assay coupled with structure-activity relationship evaluation can provide insight into MoA and impact medicinal chemistry efforts, ultimately leading to the production of inherently safer molecules.

Production of liver preneoplasia and gallbladder agenesis in turkey fetuses administered diethylnitrosamine.

The in ovo carcinogenicity assessing (IOCA) assay was used to examine the morphological changes in fetal turkey livers caused by the DNA-reactive carcinogen diethylnitrosamine (DEN). Fertilized turkey eggs were allocated into 3 groups: nondosed control (NDC), vehicle (water) control (VC) and DEN-dosed. At day 0, the fertilized eggs of the dosed groups were injected with 1 (LD) or 4 (HD) mg/egg (about 12.5 or 50 mg/kg egg) of DEN and the VC were injected with water. All eggs were allowed to incubate at 37°C and 60% humidity for 24 days. The fetal livers were collected and processed for histopathological evaluation (H&E staining). Typical survival rates were 82% for the NDC, 50% for the VC and 16-65% for the DEN-dosed fetuses. No difference in histology was found between NDC and VC control groups. Both DEN concentrations produced dose-related liver findings consisting of foci of altered hepatocytes (FAH), which had assumed a tubular cord (glandular) pattern, and in HD DEN group the FAH assumed a tumor-like morphology. In addition, the high DEN dose produced gallbladder agenesis. Thus, DEN produced both hepatocellular transformation (FAH) similar to preneoplastic microscopic changes in adult rodents, reflecting disruption of the fetal processes of differentiation and proliferation, and also teratogenicity (gallbladder agenesis).

Fluoroquinolones lower constitutive H2AX and ATM phosphorylation in TK6 lymphoblastoid cells via modulation of the intracellular redox status.

Accumulation of reactive oxygen species (ROS)-induced damage and mutations in the genomic DNA is considered the primary etiology of aging and age-related pathologies including cancer. Strategies aimed at slowing these conditions often involve protecting against oxidative DNA damage via modulation of the intracellular redox state. Recently, a biomarker of DNA double-strand breaks (DSBs), serine 139-phosphorylated histone H2AX (gammaH2AX), and its upstream mediator, activated PI-3-related kinase, ATM (ATM(P1981)), were shown to be constitutively expressed in cells and modulated by antioxidant treatment. Thus, both constitutive histone H2AX phosphorylation (CHP) and constitutive ATM activation (CAA) are thought to reflect a cell's response to endogenous ROS-induced DSBs. In the present study, we investigated the effects of a battery of fluoroquinolone (FQ) compounds, namely ciprofloxacin, enrofloxacin, gatifloxacin, lomefloxacin and ofloxacin, on CHP and CAA in human TK6 lymphoblastoid cells. All FQs tested reduced CHP and CAA compared to controls following 6 and 24 h treatment with CAA being more sensitive to their effects at both time points. In addition, intracellular ROS levels and mitochondrial activities were also lowered in FQ-treated cells at 6 and 24 h.We presume that FQs mediate this effect via a combination of ROS-scavenging and mitochondrial suppression and therefore may protect against the onset or may slow the progression of numerous oxidative pathophysiological conditions.

H2AX phosphorylation as a genotoxicity endpoint.

The gammaH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006-60 microg/ml), the alkylating agent methyl methanesulfonate (1.3-65 microg/ml) and the direct DNA-damaging agent bleomycin (0.1-10 microg/ml) all produced a positive concentration-response relationship. The non-genotoxic compounds ampicillin (0.035-3500 microg/ml) and sodium chloride (0.058-580 microg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay. These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the gammaH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median gammaH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.

Genotoxicity of topoisomerase II inhibitors: an anti-infective perspective.

At present, an inevitable consequence of a chemical's inhibitory activity on key regulators of DNA topology in bacteria, the type II topoisomerases, is a less pronounced effect on their eukaryotic counterparts. In the context of anti-infectives drug development, this may pose a risk to patient safety as inhibition of eukaryotic type II topoisomerases (TOPO II) can result in the generation of DNA double-strand breaks (DSBs), which have the potential to manifest as mutations, chromosome breakage or cell death. The biological effects of several TOPO II inhibitors in mammalian cells are described herein; their modulation of DSB damage response parameters is examined and evidence for the existence of a threshold concept for genotoxicity and its relevance in safety assessment is discussed. The potential utility of gammaH2AX, a promising and highly sensitive molecular marker for DSBs, in a novel genotoxicity 'pre-screen' to conventional assays is also highlighted.