Bumblebee nest departures under low light conditions at sunrise and sunset.

Only a few diurnal animals, such as bumblebees, extend their activity into the time around sunrise and sunset when illumination levels are low. Low light impairs viewing conditions and increases sensory costs, but whether diurnal insects use low light as a cue to make behavioural decisions is uncertain. To investigate how they decide to initiate foraging at these times of day, we observed bumblebee nest-departure behaviours inside a flight net, under naturally changing light conditions. In brighter light bees did not attempt to return to the nest and departed with minimal delay, as expected. In low light the probability of non-departures increased, as a small number of bees attempted to return after spending time on the departure platform. Additionally, in lower illumination bees spent more time on the platform before flying away, up to 68 s. Our results suggest that bees may assess light conditions once outside the colony to inform the decision to depart. These findings give novel insights into how behavioural decisions are made at the start and the end of a foraging day in diurnal animals when the limits of their vision impose additional costs on foraging efficiency.

An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

Severe Hypoglycemia and Impaired Awareness of Hypoglycemia Persist in People With Type 1 Diabetes Despite Use of Diabetes Technology: Results From a Cross-sectional Survey.

OBJECTIVE: To determine how diabetes technologies, including continuous glucose monitoring (CGM) and automated insulin delivery (AID) systems, impact glycemic metrics, prevalence of severe hypoglycemic events (SHEs), and impaired awareness of hypoglycemia (IAH) in people with type 1 diabetes in a real-world setting within the U.S. RESEARCH DESIGN AND METHODS: In this retrospective, observational study with cross-sectional elements, participants aged ≥18 years were enrolled from the T1D Exchange Registry/online community. Participants completed a one-time online survey describing glycemic metrics, SHEs, and IAH. The primary objective was to determine the proportions of participants who reported achieving glycemic targets (assessed according to self-reported hemoglobin A1c) and had SHEs and/or IAH. We performed additional subgroup analyses focusing on the impact of CGM and insulin delivery modality. RESULTS: A total of 2,074 individuals with type 1 diabetes were enrolled (mean ± SD age 43.0 ± 15.6 years and duration of type 1 diabetes 26.3 ± 15.3 years). The majority of participants (91.7%) were using CGM, with one-half (50.8%) incorporating AID. Despite high use of diabetes technologies, only 57.7% reported achieving glycemic targets (hemoglobin A1c <7%). SHEs and IAH still occurred, with ∼20% of respondents experiencing at least one SHE within the prior 12 months and 30.7% (95% CI 28.7, 32.7) reporting IAH, regardless of CGM or AID use. CONCLUSIONS: Despite use of advanced diabetes technologies, a high proportion of people with type 1 diabetes do not achieve glycemic targets and continue to experience SHEs and IAH, suggesting an ongoing need for improved treatment strategies.

Perceptions of Continuous Glucose Monitoring Systems in the T1D Exchange Diabetes Registry: Satisfaction, Concerns, and Areas for Future Improvement.

Manufacturers continue to improve performance and usability of continuous glucose monitoring (CGM) systems. As CGM becomes a standard of care, especially for people on insulin therapy, it is important to routinely gauge how satisfied people with diabetes are with this technology. This article describes survey feedback from a large cohort of people with diabetes using older and current CGM systems and highlights areas of current satisfaction, concern, and future system improvement.

Multi-endpoint analysis of cadmium chloride-induced genotoxicity shows role for reactive oxygen species and p53 activation in DNA damage induction, cell cycle irregularities, and cell size aberrations.

Cadmium chloride (CdCl2) is a known genotoxic carcinogen, with a mechanism of action thought to partly involve the generation of reactive oxygen species (ROS). We applied here a multi-endpoint approach in vitro to explore the impact of CdCl2 on both the genome and on wider cell biology pathways relevant to cancer. Multi-endpoint approaches are believed to offer greater promise in terms of understanding the holistic effects of carcinogens in vitro. This richer understanding may help better classification of carcinogens as well as allowing detailed mechanisms of action to be identified. We found that CdCl2 caused DNA damage [micronuclei (MN)] in both TK6 and NH32 cells in a dose-dependent manner after 4 h exposure (plus 23 h recovery), with lowest observable effect levels (LOELs) for MN induction of 1 µM (TK6) and 1.6 µM (NH32). This DNA damage induction in TK6 cells was ROS dependent as pretreatment with the antioxidant N-Acetyl Cysteine (1 mM), abrogated this effect. However, 2',7'-dichlorofluorescin diacetate was not capable of detecting the ROS induced by CdCl2. The use of NH32 cells allowed an investigation of the role of p53 as they are a p53 null cell line derived from TK6. NH32 showed a 10-fold increase in MN in untreated cells and a similar dose-dependent effect after CdCl2 treatment. In TK6 cells, CdCl2 also caused activation of p53 (accumulation of total and phosphorylated p53), imposition of cell cycle checkpoints (G2/M) and intriguingly the production of smaller and more eccentric (elongated) cells. Overall, this multi-endpoint study suggests a carcinogenic mechanism of CdCl2 involving ROS generation, oxidative DNA damage and p53 activation, leading to cell cycle abnormalities and impacts of cell size and shape. This study shows how the integration of multiple cell biology endpoints studied in parallel in vitro can help mechanistic understanding of how carcinogens disrupt normal cell biology.

Severe Hypoglycemia and the Use of Glucagon Rescue Agents: An Observational Survey in Adults With Type 1 Diabetes.

Severe hypoglycemia (SH) is the most frequent and potentially serious complication affecting individuals with type 1 diabetes and can have major clinical and psychosocial consequences. Glucagon is the only approved treatment for SH that can be administered by non-health care professionals (HCPs); however, reports on the experiences and emotions of people with type 1 diabetes associated with SH and glucagon rescue use are limited. This survey study demonstrated that an increasing number of individuals with type 1 diabetes have current and filled prescriptions for glucagon and have been educated about glucagon rescue use by an HCP. Despite this positive trend, challenges with SH remain, including a high level of health care resource utilization, considerable out-of-pocket expenses for glucagon kits, a high prevalence of hypoglycemia unawareness, and a negative emotional impact on individuals with diabetes. Nocturnal and exercise-related hypoglycemia were concerns for most survey participants.

Prevalence of fear of hypoglycemia in adults with type 1 diabetes using a newly developed screener and clinician's perspective on its implementation.

INTRODUCTION: Fear of hypoglycemia (FoH) affects quality of life, emotional well-being, and diabetes management among people with type 1 diabetes (PwT1D). American Diabetes Association's (ADA) guidelines recommend assessing FoH in clinical practice. However, existing FoH measures are commonly used in research and not in clinical practice. In this study, prevalence of FoH was assessed in PwT1D using a newly developed FoH screener for clinical practice; its association with established measures and outcomes was also determined. In addition, healthcare providers' (HCPs) perspectives on implementing FoH screener into real-world practice were explored. RESEARCH DESIGN AND METHODS: This multiphase observational study used mixed methods in two phases. First, we collected a cross-sectional survey (including the screener) from PwT1D (≥18 years) from T1D Exchange Quality Improvement Collaborative adult clinics. Pearson correlations and regression analyses were performed on diabetes outcome measures using screener scores. Second, we conducted focus groups among HCPs who treat PwT1D and descriptive analysis to summarize results. RESULTS: We included 553 PwT1D. Participants had a mean±SD age of 38.9±14.2 years and 30% reported a high FoH total score. Regression analyses showed that higher A1c and higher number of comorbidities were significantly associated with high FoH (p<0.001). High FoH worry and behavior scores were significantly associated with 8-Item Patient Health Questionnaire and 7-Item Generalized Anxiety Disorder Scale scores. Participants with ≥1 severe hypoglycemia event(s) and impaired awareness of hypoglycemia had higher odds of high FoH. Eleven HCPs participated in focus group interviews; they expressed that the FoH screener is clinically necessary and relevant but poses implementation challenges that must be addressed. CONCLUSIONS: Our results demonstrate FoH is common in PwT1D and affects their psychosocial well-being and diabetes management. In alignment with ADA position statement, HCP focus group results emphasize importance of screening for FoH. Implementing this newly developed FoH screener may help HCPs identify FoH in PwT1D.

Emerging Adult and Caregiver Psychosocial Experiences with Severe Hypoglycemic Events and the Perceived Impact of Nasal Glucagon: A Cross-Sectional Study.

INTRODUCTION: Severe hypoglycemic events are distressing. Although past studies have shown that young adulthood is a potentially distressing time, few studies have explored distress about severe hypoglycemia in this age group. The real-world psychosocial experiences of potential severe hypoglycemic events and the perceived impact of glucagon treatments like nasal glucagon are currently unknown. We explored perceptions of severe hypoglycemic events and impact of nasal glucagon on psychosocial experiences with these events in emerging adults with type 1 diabetes and caregivers of emerging adults and children/teens. Further, we compared perceptions of preparedness and protection in handling severe hypoglycemic events with nasal glucagon versus the emergency glucagon kit that requires reconstitution (e-kit). METHODS: This observational, cross-sectional study enrolled emerging adults (aged 18-26; N = 364) with type 1 diabetes, caregivers of emerging adults (aged 18-26; N = 138) with type 1 diabetes, and caregivers of children/teens (aged 4-17; N = 315) with type 1 diabetes. Participants completed an online survey about their experiences with severe hypoglycemia, perceptions of nasal glucagon impact on psychosocial experiences, and perceptions of feeling prepared and protected with nasal glucagon and the e-kit. RESULTS: Many emerging adults (63.7%) agreed that the experience of severe hypoglycemic events was distressing; 33.3% and 46.7% of caregivers of emerging adults and children/teens, respectively, reported distress. Participants reported positive perceptions of nasal glucagon impact, particularly improved confidence in other people's ability to help during severe hypoglycemic events: emerging adults, 81.4%; caregivers of emerging adults, 77.6%; caregivers of children/teens, 75.5%. Participants demonstrated higher perceptions of preparedness and protection for nasal glucagon than for the e-kit (p < 0.001). CONCLUSIONS: Participants reported improved confidence in other people's ability to help during severe hypoglycemic events since having nasal glucagon available. This suggests that nasal glucagon may help broaden the support network for young people with type 1 diabetes and their caregivers.

Implementation of a knowledge-based decision support system for treatment plan auditing through automation.

BACKGROUND: Independent auditing is a necessary component of a comprehensive quality assurance (QA) program and can also be utilized for continuous quality improvement (QI) in various radiotherapy processes. Two senior physicists at our institution have been performing a time intensive manual audit of cross-campus treatment plans annually, with the aim of further standardizing our planning procedures, updating policies and guidelines, and providing training opportunities of all staff members. PURPOSE: A knowledge-based automated anomaly-detection algorithm to provide decision support and strengthen our manual retrospective plan auditing process was developed. This standardized and improved the efficiency of the assessment of our external beam radiotherapy (EBRT) treatment planning across all eight campuses of our institution. METHODS: A total of 843 external beam radiotherapy plans for 721 lung patients from January 2020 to March 2021 were automatically acquired from our clinical treatment planning and management systems. From each plan, 44 parameters were automatically extracted and pre-processed. A knowledge-based anomaly detection algorithm, namely, "isolation forest" (iForest), was then applied to the plan dataset. An anomaly score was determined for each plan using recursive partitioning mechanism. Top 20 plans ranked with the highest anomaly scores for each treatment technique (2D/3D/IMRT/VMAT/SBRT) including auto-populated parameters were used to guide the manual auditing process and validated by two plan auditors. RESULTS: The two auditors verified that 75.6% plans with the highest iForest anomaly scores have similar concerning qualities that may lead to actionable recommendations for our planning procedures and staff training materials. The time to audit a chart was approximately 20.8 min on average when done manually and 14.0 min when done with the iForest guidance. Approximately 6.8 min were saved per chart with the iForest method. For our typical internal audit review of 250 charts annually, the total time savings are approximately 30 hr per year. CONCLUSION: iForest effectively detects anomalous plans and strengthens our cross-campus manual plan auditing procedure by adding decision support and further improve standardization. Due to the use of automation, this method was efficient and will be used to establish a standard plan auditing procedure, which could occur more frequently.

Development and validation of fear of hypoglycemia screener: results from the T1D exchange registry.

BACKGROUND: Fear of Hypoglycemia (FoH) in people with diabetes has a significant impact on their quality of life, psychological well-being, and self-management of disease. There are a few questionnaires assessing FoH in people living with diabetes, but they are more often used in research than clinical practice. This study aimed to develop and validate a short and actionable FoH screener for adults living with type 1 diabetes (T1D) for use in routine clinical practice. METHODS: We developed an initial screener based on literature review and, interviews with healthcare providers (HCPs) and people with T1D. We developed a cross-sectional web-based survey, which was then conducted to examine the reliability and validity of the screener. Adults (aged ≥ 18 years) with diagnosis of T1D for ≥ 1 year were recruited from the T1D Exchange Registry (August-September 2020). The validation analyses were conducted using exploratory factor analyses, correlation, and multivariable regression models for predicting cut-off scores for the final screener. RESULTS: The final FoH screener comprised nine items assessing two domains, "worry" (6-items) and "avoidance behavior" (three items), in 592 participants. The FoH screener showed good internal consistency (Cronbach's α = 0.88). The screener also demonstrated high correlations (r = 0.71-0.75) with the Hypoglycemia Fear Survey and moderate correlations with depression, anxiety, and diabetes distress scales (r = 0.44-0.66). Multivariable regression analysis showed that higher FoH screener scores were significantly associated with higher glycated hemoglobin (HbA1c) (b = 0.04) and number of comorbidities (b = 0.03). CONCLUSIONS: This short FoH screener demonstrated good reliability and validity. Further research is planned to assess clinical usability to identify patients with FoH and assist effective HCP-patient conversations.

Evaluation of endpoints for the study and diagnosis of mitochondrial toxicity and disease: a narrative review.

Mitochondrial DNA mutation and toxicity have been linked to several inherited and acquired diseases; however, these are challenging to diagnose and characterize due to clinical and genetic heterogeneity. This review investigates current techniques for the analysis of mitochondrial perturbations, and novel, emerging endpoints for routine application within the clinical setting. Particular focus is given to the biochemistry of the mitochondria influencing each endpoint and the relation of these to toxicity. Current approaches such as the use of metabolic markers (e.g. lactate production), and muscle biopsies to measure mitochondrial proteins were found to lack specificity. Newly emerging identified endpoints were: fibroblast growth factor-21, glucose uptake, mitochondrial membrane potential, mitochondrial morphology, mtDNA heteroplasmy, and mutation of mtDNA and nuclear DNA. Owed to the advancement in genetic analysis techniques, it is suggested by this review that genotypic endpoints of mtDNA mutation and heteroplasmy show particular promise as indicators of mitochondrial disease. It is, however, acknowledged that any single endpoint in isolation offers limited information; therefore, it is recommended that analysis of several endpoints simultaneously will offer the greatest benefit in terms of disease diagnosis and study. It is hoped that this review further highlights the need for advancement in understanding mitochondrial disease.

Limiting motorboat noise on coral reefs boosts fish reproductive success.

Anthropogenic noise impacts are pervasive across taxa, ecosystems and the world. Here, we experimentally test the hypothesis that protecting vulnerable habitats from noise pollution can improve animal reproductive success. Using a season-long field manipulation with an established model system on the Great Barrier Reef, we demonstrate that limiting motorboat activity on reefs leads to the survival of more fish offspring compared to reefs experiencing busy motorboat traffic. A complementary laboratory experiment isolated the importance of noise and, in combination with the field study, showed that the enhanced reproductive success on protected reefs is likely due to improvements in parental care and offspring length. Our results suggest noise mitigation could have benefits that carry through to the population-level by increasing adult reproductive output and offspring growth, thus helping to protect coral reefs from human impacts and presenting a valuable opportunity for enhancing ecosystem resilience.

Emergency Glucagon: a Focused Review of Psychosocial Experiences of Rescue Drugs for Type 1 Diabetes.

PURPOSE OF REVIEW: The purpose of this paper is to describe rescue glucagon types, safety, efficacy, and preferences, as well as to review articles regarding emergency glucagon usage, severe hypoglycemia, and the emotions of both phenomena. We conducted a review of current literature on glucagon usage and the emotional impact of severe hypoglycemia on people with diabetes (PwD) and the caregivers of people with type 1 diabetes (T1D). RECENT FINDINGS: Minimal research exists pertaining to glucagon and severe hypoglycemic experiences in PwD, which is troubling considering the severity of risks and possible side effects. Recent articles described negative emotions such as fear, anxiety, stress, helplessness, shame, embarrassment, loneliness, frustration, hopefulness, and uncertainty surrounding glucagon usage. There is scarce research regarding PwD's emotions surrounding severe hypoglycemia and rescue glucagon use. Additional research is needed to investigate the emotions and feelings people with T1D and their caregivers' experience pertaining to severe hypoglycemia and emergency glucagon use.

Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro.

Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both ∼16 nm (4.34 × 10-3 to 17.36 × 10-3 mg/mL) Gold (Au) and ∼14 nm (0.85 × 10-5 to 3.32 × 10-5 mg/mL) Silver (Ag) ENPs upon two commonly used lung epithelial cell lines, 16HBE14o- and A549. Following cytotoxicity analysis (via Trypan Blue and Lactate Dehydrogenase assay), two sub-lethal concentrations were selected for genotoxicity analysis using the cytokinesis-blocked micronucleus assay. Whilst both ENP types induced significant oxidative stress, Ag ENPs (1.66 × 10-5 mg/mL) did not display a significant genotoxic response in either epithelial cell lines, but Au ENPs (8.68 × 10-3 mg/mL) showed a highly significant 2.63-fold and 2.4-fold increase in micronucleus frequency in A549 and 16HBE14o- cells respectively. It is hypothesized that the DNA damage induced by acute 24-h Au ENP exposure resulted in a cell cycle stall indicated by the increased mononuclear cell fraction (>6.0-fold) and cytostasis level. Albeit insignificant, a small reduction in telomere length was observed following acute exposure to both ENPs which could indicate the potential for ENP mediated telomere attrition. Finally, from the data shown, both in vitro lung cell cultures (16HBE14o- and A549) are equally as suitable and reliable for the in vitro ENP hazard identification approach adopted in this study.

"It's embarrassing. I get angry. I get frustrated.": Understanding severe hypoglycemia and glucagon usage from the perspectives of people with type 1 diabetes.

Introduction: This study characterized the emotional impact of severe hypoglycemia, views of glucagon, and barriers to glucagon use from the perspective of adults with type 1 diabetes (T1D). Methods: Participants included individuals recruited from the T1D Exchange online community. The current study conducted 7 focus groups consisting of adults with T1D (N = 38, average age 49.4, SD = 16.11 years). Average duration of diabetes was 34.4 years (SD = 17.3) and average self-reported A1c was 6.8 % (SD = 0.7). Focus group interviews were recorded, transcribed, and thematically analyzed. Results: A range of emotions was expressed about severe hypoglycemia including fear, anxiety, stress, frustration, shame, and embarrassment. Participants frequently identified prescription cost and insurance deductibles as barriers to glucagon use. Participants were also concerned about ease of administration-how difficult it is to prepare the glucagon in an emergency. Many participants expressed a preference for auto-injectables over nasal administration. Timing of glucagon action and time to recovery were high priorities. Some participants, while they had not self-administered glucagon, were interested in a mini-dose glucagon they could self-administer. They also identified desirable characteristics of glucagon treatment including reduced cost, long shelf-life, and quick activation. Conclusions: These results highlight the attitudes about severe hypoglycemia and emergency treatment with glucagon. Healthcare professionals should assess glucagon training needs and knowledge when they meet with their patients with diabetes.

Attack behaviour in naive gyrfalcons is modelled by the same guidance law as in peregrine falcons, but at a lower guidance gain.

The aerial hunting behaviours of birds are strongly influenced by flight morphology and ecology, but little is known of how this relates to the behavioural algorithms guiding flight. Here, we used GPS loggers to record the attack trajectories of captive-bred gyrfalcons (Falco rusticolus) during their maiden flights against robotic aerial targets, which we compared with existing flight data from peregrine falcons (Falco peregrinus). The attack trajectories of both species were well modelled by a proportional navigation (PN) guidance law, which commands turning in proportion to the angular rate of the line-of-sight to target, at a guidance gain N However, naive gyrfalcons operate at significantly lower values of N than peregrine falcons, producing slower turning and a longer path to intercept. Gyrfalcons are less manoeuvrable than peregrine falcons, but physical constraint is insufficient to explain the lower values of N we found, which may reflect either the inexperience of the individual birds or ecological adaptation at the species level. For example, low values of N promote the tail-chasing behaviour that is typical of wild gyrfalcons and which apparently serves to tire their prey in a prolonged high-speed pursuit. Likewise, during close pursuit of typical fast evasive prey, PN will be less prone to being thrown off by erratic target manoeuvres at low guidance gain. The fact that low-gain PN successfully models the maiden attack flights of gyrfalcons suggests that this behavioural algorithm is embedded in a guidance pathway ancestral to the clade containing gyrfalcons and peregrine falcons, though perhaps with much deeper evolutionary origins.

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

The effect of chronic dosing and p53 status on the genotoxicity of pro-oxidant chemicals in vitro.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2',7'-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.