Circadian effects on UV-induced damage and mutations.

Skin cancer is the most diagnosed type of cancer in the United States, and while most of these malignancies are highly treatable, treatment costs still exceed $8 billion annually. Over the last 50 years, the annual incidence of skin cancer has steadily grown; therefore, understanding the environmental factors driving these types of cancer is a prominent research-focus. A causality between ultraviolet radiation (UVR) exposure and skin cancer is well-established, but exposure to UVR alone is not necessarily sufficient to induce carcinogenesis. The emerging field of circadian biology intersects strongly with the physiological systems of the mammalian body and introduces a unique opportunity for analyzing mechanisms of homeostatic disruption. The circadian clock refers to the approximate 24-hour cycle, in which protein levels of specific clock-controlled genes (CCGs) fluctuate based on the time of day. Though these CCGs are tissue specific, the skin has been observed to have a robust circadian clock that plays a role in its response to UVR exposure. This in-depth review will detail the mechanisms of the circadian clock and its role in cellular homeostasis. Next, the skin's response to UVR exposure and its induction of DNA damage and mutations will be covered - with an additional focus placed on how the circadian clock influences this response through nucleotide excision repair. Lastly, this review will discuss current models for studying UVR-induced skin lesions and perturbations of the circadian clock, as well as the impact of these factors on human health.

Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms.

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.

Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks.

Infant acute leukemias are aggressive and characterized by rapid onset after birth. The majority harbor translocations involving the MLL gene with AF9 as one of its most common fusion partners. MLL and AF9 loci contain breakpoint cluster regions (bcrs) with sequences hypothesized to be targets of topoisomerase II inhibitors that promote translocation formation. Overlap of MLL bcr sequences associated with both infant acute leukemia and therapy-related leukemia following exposure to the topoisomerase II inhibitor etoposide led to the hypothesis that exposure during pregnancy to biochemically similar compounds may promote infant acute leukemia. We established a reporter system to systematically quantitate and stratify the potential for such compounds to promote chromosomal translocations between the MLL and AF9 bcrs analogous to those in infant leukemia. We show bioflavonoids genistein and quercetin most biochemically similar to etoposide have a strong association with MLL-AF9 bcr translocations, while kaempferol, fisetin, flavone, and myricetin have a weak but consistent association, and other compounds have a minimal association in both embryonic stem (ES) and hematopoietic stem cell (HSC) populations. The frequency of translocations induced by bioflavonoids at later stages of myelopoiesis is significantly reduced by more than one log. The MLL and AF9 bcrs are sensitive to these agents and recombinogenic independent of their native context suggesting bcr sequences themselves are drivers of illegitimate DNA repair reactions and translocations, not generation of functional oncogenic fusions. This system provides for rapid systematic screening of relative risk, dose dependence, and combinatorial impact of multitudes of dietary and environmental exposures on MLL-AF9 translocations. Environ. Mol. Mutagen. 60: 154-167, 2019. © 2018 Wiley Periodicals, Inc.

Effect of Systemic Triphenylphosphonium on Organ Function and Oxidative Stress.

Conditions of systemic stress can lead to increased reactive oxygen species production, mitochondrial dysfunction, systemic inflammation, and multiorgan dysfunction. Triphenylphosphonium (TPP+) is a lipophilic cation used to target therapeutics to mitochondria. We sought to determine the effects of TPP+ on mitochondrial integrity. Male rats were anesthetized and TPP+ (5 mg/kg) or vehicle (saline) was administered intravenously 30-minutes after anesthesia initiation and intraperitoneally (20 mg/kg) 60-minutes later. Rats were exsanguinated 2-hours postinjection. Cardiac, pulmonary, hepatic, splenic, and renal tissues were analyzed for inflammation, lipid peroxidation, endogenous antioxidant activity, cytokine expression, and mitochondrial function. In vitro modeling was performed using freshly isolated hepatocytes subjected to 8-hours hypoxia/30-minutes reoxygenation in the absence or presence of TPP+. TPP+ increased lipid peroxidation in the liver, lung, and kidney as well as antioxidant activity in the liver, kidney, and spleen. Conversely, antioxidant activity decreased in the lung with TPP+. In addition, TPP+ altered hepatic inflammatory mediators. In vitro, TPP+ attenuated oxygen consumption and, when combined with hypoxic injury, depolarized mitochondrial membranes in hepatocytes. TPP+ induces systemic responses associated with oxidative stress and worsening pathologies in animals. Caution should be exercised when employing TPP+ for therapeutics.

Cytochrome c limits oxidative stress and decreases acidosis in a rat model of hemorrhagic shock and reperfusion injury.

BACKGROUND: Hemorrhagic shock and reperfusion (HSR) injury leads to a cascade of reactive oxygen species (ROS) production and mitochondrial dysfunction, which results in energy failure, cell death, and multiple organ dysfunction. Cytochrome c (cyt c) is the final electron carrier in the mitochondrial electron transport chain providing the electrochemical force for ATP production. We sought to determine whether exogenous cyt c administration would improve parameters of organ dysfunction and/or mitochondrial stability in a rat model of HSR. METHODS: Male rats were hemorrhaged to a mean arterial pressure (MAP) of 33 ± 2.0 mm Hg for 1 hour before resuscitation. Saline or cyt c (0.8 mg [HSR-LoCC] or 3.75 mg [HSR-HiCC]) was administered (i.v.) 30 minutes before resuscitation. Rats were euthanized by cardiac puncture 2 hours post-surgery and tissue collected and analyzed for lipid peroxidation, endogenous antioxidant activity (glutathione peroxidase (GPx) and catalase), TNF-α expression, mitochondrial function (complex-I activity), and circulating mitochondrial DNA (mtDNA). RESULTS: Cyt c administration improved lactate clearance, decreased hepatic lipid peroxidation, increased hepatic GPx activity, restored pulmonary TNF-α to sham activity levels, and increased hepatic complex-I activity. Furthermore, addition of exogenous cyt c decreased circulating levels of mtDNA. CONCLUSIONS: These studies demonstrate that cyt c reduces markers of physiologic stress, decreases oxidative stress, and lowers levels of circulating mtDNA. The impact of cytochrome c is organ specific. Further studies remain to determine the sum of the effects of cytochrome c on overall outcome.

Examining the role of follow-up skeletal surveys in non-accidental trauma.

BACKGROUND: Diagnosing NAT (non-accidental trauma) includes a skeletal survey to identify injuries. A follow-up survey is performed for missed injuries. This study examines the necessity of follow-up surveys. METHODS: The trauma database identified cases of suspected NAT in <4 years olds (2013-2014). Data were stratified by survey, age, injury, then analyzed for the prevalence of findings. All analyses (relative risk, prevalence and odds ratios) utilized RealStats Resource Pack (Trento, Italy). RESULTS: 79% positive initial findings and no new follow up findings. Those with negative initial imaging, had no missed injuries. Initial scans were 27.6X more likely to be positive. Fractured skull (31.3), femur (17.2) and ribs (15.7) were the most prevalent. No pelvic fractures and <1% spinal injuries despite both having the greatest radiation exposure. Repeat scans rarely identify findings for age >12 months. CONCLUSIONS: Follow-up skeletal surveys maybe unnecessary without clinical evidence. Uncommon pelvic and spinal fractures may warrant exclusion from surveys unless clinically indicated.