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Adam J Kruse,1 Andrew S Nugent,2 Andrew R Peterson3 1Carver College of Medicine, The University of Iowa, Iowa City, IA, USA; 2Department of Emergency Medicine, The University of Iowa, Iowa City, IA, USA; 3Department of Pediatrics, The University of Iowa, Iowa City, IA, USA Purpose: Traumatic brain injury (TBI) is a significant cause of death and disability in the United States. Many patients with TBI are initially treated in the emergency department (ED), but there is no evidence-based method of detecting or grading TBI in patients who have normal structural neuroimaging. This study aims to evaluate the validity of two common sideline concussion tests. The Concussion Symptom Severity Score (CSSS) and modified Balance Error Scoring System (mBESS) tests are well-validated sideline tests for concussion, but have not been validated in the setting of non-sport-related concussion, in settings other than the sideline or athletic training room or in moderate or severe TBI. Patients and methods: One hundred forty-eight subjects who had sustained a TBI within the previous 72 hours and 53 healthy control subjects were enrolled. CSSS and mBESS were administered. Clinical outcomes were followed up prospectively. Results: The CSSS was collected in 147 TBI subjects but only 51 TBI subjects were able to complete the mBESS. The CSSS was collected for all 53 control subjects, and the mBESS was completed for 51 control subjects. The mean CSSS for TBI and control subjects was 32.25 and 2.70, respectively (P < 0.001). The average mBESS for TBI and control subjects was 7.43 and 7.20, respectively (P = 0.82). CSSS greater than 5.17 was 93.43% sensitive and 69.84% specific for TBI. Conclusion: The mBESS is poorly tolerated and, among those who can complete the test, not sensitive to TBI in the ED. The CSSS is both sensitive to TBI and well tolerated. Keywords: traumatic brain injury, balance, emergency room, Sport Concussion Assessment Tool, concussion symptom severity score, modified balance error scoring system

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Dysregulation of glutamate transporters has been a common finding across animal models of epilepsy and in patients with TLE. In this study, we investigate NRG-1/ErbB4 signaling in epileptogenesis and the neuroprotective effects of NRG-1 treatment in a mouse model of temporal lobe epilepsy. Using immunohistochemistry, we report the first evidence for NRG-1/ErbB4-dependent selective upregulation of glutamate transporter EAAC1 and bihemispheric neuroprotection by exogeneous NRG-1 in the intrahippocampal kainic acid (IHKA) model of TLE. Our findings provide evidence that dysregulation of glutamate transporter EAAC1 contributes to the development of epilepsy and can be therapeutically targeted to reduce neuronal death following IHKA-induced status epilepticus (SE)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Astrocytic glutamate transporters are crucial for glutamate homeostasis in the brain, and dysregulation of these transporters can contribute to the development of epilepsy. Glutamate transporter-1 (GLT-1) is responsible for the majority of glutamate uptake in the dorsal forebrain and has been shown to be reduced at epileptic foci in patients and preclinical models of temporal lobe epilepsy (TLE). Current antiepileptic drugs (AEDs) work primarily by targeting neurons directly through suppression of excitatory neurotransmission or enhancement of inhibitory neurotransmission, which can lead to both behavioral and psychiatric side effects. This study investigates the therapeutic capacity of astrocyte-specific AAV-mediated GLT-1 expression in the intrahippocampal kainic acid (IHKA) model of TLE. In this study, we used Western blot analysis, immunohistochemistry, and long-term-video EEG monitoring to demonstrate that cell-type-specific upregulation of GLT-1 in astrocytes is neuroprotective at early time points during epileptogenesis, reduces seizure frequency and total time spent in seizures, and eliminates large behavioral seizures in the IHKA model of epilepsy. Our findings suggest that targeting glutamate uptake is a promising therapeutic strategy for the treatment of epilepsy., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background Thoracic aortic aneurysms (TAAs) occur because of abnormal remodeling of aortic extracellular matrix and are accompanied by the emergence of proteolytically active myofibroblasts. The microRNA miR-133a regulates cellular phenotypes and is reduced in clinical TAA specimens. This study tested the hypothesis that miR-133a modulates aortic fibroblast phenotype, and overexpression by lentivirus attenuates the development of TAA in a murine model. Methods and Results TAA was induced in mice. Copy number of miR-133a was reduced in TAA tissue and linear regression analysis confirmed an inverse correlation between aortic diameter and miR-133a. Analyses of phenotypic markers revealed an mRNA expression profile consistent with myofibroblasts in TAA tissue. Fibroblasts were isolated from the thoracic aortae of mice with/without TAA. When compared with controls, miR-133a was reduced, migration was increased, adhesion was reduced, and the ability to contract a collagen disk was increased. Overexpression/knockdown of miR-133a controlled these phenotypes. After TAA induction in mice, a single tail-vein injection of either miR-133a overexpression or scrambled sequence (control) lentivirus was performed. Overexpression of miR-133a attenuated TAA development. The pro-protein convertase furin was confirmed to be a target of miR-133a by luciferase reporter assay. Furin was elevated in this murine model of TAA and repressed by miR-133a replacement in vivo resulting in reduced proteolytic activation. Conclusions miR-133a regulates aortic fibroblast phenotype and over-expression prevented the development of TAA in a murine model. These findings suggest that stable alterations in aortic fibroblasts are associated with development of TAA and regulation by miR-133a may lead to a novel therapeutic strategy.

Astrocytes regulate and respond to extracellular glutamate levels in the central nervous system (CNS) via the Na + -dependent glutamate transporters glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST) and the metabotropic glutamate receptors (mGluR) 3 and mGluR5. Both impaired astrocytic glutamate clearance and changes in metabotropic glutamate signaling could contribute to the development of epilepsy. Dysregulation of astrocytic glutamate transporters, GLT-1 and GLAST, is a common finding across patients and preclinical seizure models. Astrocytic metabotropic glutamate receptors, particularly mGluR5, have been shown to be dysregulated in both humans and animal models of temporal lobe epilepsy (TLE). In this review, we synthesize the available evidence regarding astrocytic glutamate homeostasis and astrocytic mGluRs in the development of epilepsy. Modulation of astrocyte glutamate uptake and/or mGluR activation could lead to novel glial therapeutics for epilepsy., (Copyright © 2020 Peterson and Binder.)

Astrocytes regulate extracellular glutamate homeostasis in the central nervous system through the Na + -dependent glutamate transporters glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST). Impaired astrocyte glutamate uptake could contribute to the development of epilepsy but the regulation of glutamate transporters in epilepsy is not well understood. In this study, we investigate the expression of GLT-1 and GLAST in the mouse intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used immunohistochemistry, synaptosomal fractionation and Western blot analysis at 1, 3, 7 and 30 days post-IHKA induced status epilepticus (SE) to examine changes in GLT-1 and GLAST immunoreactivity and synaptosomal expression during the development of epilepsy. We found a significant upregulation in GLT-1 immunoreactivity at 1 and 3 days post-IHKA in the ipsilateral dorsal hippocampus. However, GLT-1 immunoreactivity and synaptosomal protein levels were significantly downregulated at 7 days post-IHKA in the ipsilateral hippocampus, a time point corresponding to the onset of spontaneous seizures in this model. GLAST immunoreactivity was increased in specific layers at 1 and 3 days post-IHKA in the ipsilateral hippocampus. GLAST synaptosomal protein levels were significantly elevated at 30 days compared to 7 days post-IHKA in the ipsilateral hippocampus. Our findings suggest that astrocytic glutamate transporter dysregulation could contribute to the development of epilepsy., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Glutamate transporter-1 (GLT-1) is a Na + -dependent transporter that plays a key role in glutamate homeostasis by removing excess glutamate in the central nervous system (CNS). GLT-1 dysregulation occurs in various neurological diseases including Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and epilepsy. Downregulation or dysfunction of GLT-1 has been a common finding across these diseases but how this occurs is still under investigation. This review aims to highlight post-translational regulation of GLT-1 which leads to its downregulation including sumoylation, palmitoylation, nitrosylation, ubiquitination, and subcellular localization. Various therapeutic interventions to restore GLT-1, their proposed mechanism of action and functional effects will be examined as potential treatments to attenuate the neurological symptoms associated with loss or downregulation of GLT-1.

Blood flow restriction (BFR) training is commonly used to induce muscle hypertrophy and strength and is gaining popularity as a modality for treatment of musculoskeletal conditions and postoperative rehabilitation. This article reviews the current literature on BFR as a rehabilitative tool, identifies gaps in the literature, and reports the authors experience using BFR in the postoperative environment.

Context:: Infections are common in contact sports. This review aims to describe the epidemiology, presenting signs and symptoms, treatment guidelines, and regulations for several common infections seen in contact sport athletes. The conditions discussed include bacterial skin infections, herpes simplex virus, molluscum contagiosum, common warts, tinea, scabies, head lice, conjunctivitis, human immunodeficiency virus, hepatitis C virus, and vaccine-preventable illnesses., Evidence Acquisition:: Searches were performed across PubMed and MEDLINE research databases. In addition, general internet search engine results and reviews of reference lists of relevant papers were used to identify additional sources of evidence., Study Design:: Clinical review., Level of Evidence:: Level 4., Results:: The most common infections seen in contact sport athletes include bacterial skin infections, herpes simplex virus, molluscum contagiosum, common warts, tinea, scabies, head lice, conjunctivitis, and vaccine-preventable illnesses. Other infections, including human immunodeficiency virus and hepatitis C, are uncommon but potentially life threatening., Conclusion:: Infections are common in contact sport athletes. The provider who cares for these athletes should be aware of the most common infections and their appropriate management. Early diagnosis and appropriate clinical management are important for treating the infected athlete, minimizing risk of transmission, minimizing time lost from competition, and preventing large outbreaks.

Background: There are approximately 2.8 million youth football players between the ages of 7 and 14 years in the United States. Rates of injury in this population are poorly described. Recent studies have reported injury rates between 2.3% and 30.4% per season and between 8.5 and 43 per 1000 exposures., Hypothesis: Youth flag football has a lower injury rate than youth tackle football. The concussion rates in flag football are lower than in tackle football., Study Design: Cohort study; Level of evidence, 3., Methods: Three large youth (grades 2-7) football leagues with a total of 3794 players were enrolled. Research personnel partnered with the leagues to provide electronic attendance and injury reporting systems. Researchers had access to deidentified player data and injury information. Injury rates for both the tackle and flag leagues were calculated and compared using Poisson regression with a log link. The probability an injury was severe and an injury resulted in a concussion were modeled using logistic regression. For these 2 responses, best subset model selection was performed, and the model with the minimum Akaike information criterion value was chosen as best. Kaplan-Meier curves were examined to compare time loss due to injury for various subgroups of the population. Finally, time loss was modeled using Cox proportional hazards regression models., Results: A total of 46,416 exposures and 128 injuries were reported. The mean age at injury was 10.64 years. The hazard ratio for tackle football (compared with flag football) was 0.45 (95% CI, 0.25-0.80; P = .0065). The rate of severe injuries per exposure for tackle football was 1.1 (95% CI, 0.33-3.4; P = .93) times that of the flag league. The rate for concussions in tackle football per exposure was 0.51 (95% CI, 0.16-1.7; P = .27) times that of the flag league., Conclusion: Injury is more likely to occur in youth flag football than in youth tackle football. Severe injuries and concussions were not significantly different between leagues. Concussion was more likely to occur during games than during practice. Players in the sixth or seventh grade were more likely to suffer a concussion than were younger players., Competing Interests: The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.

Helping a wrestler manage body weight can be a daunting process for a pediatric health care provider. Each high school wrestling program has been mandated by the National Federation of State High School Associations to determine an appropriate weight classification for each individual wrestler. This article discusses how an appropriate weight class is determined, the methods for ascertaining a person's hydration status and body density, and the importance of a fully hydrated and normally nourished state that will allow for optimal athletic performance for a wrestler., (Copyright 2016, SLACK Incorporated.)

Lactic acid has played an important role in the traditional theory of muscle fatigue and limitation of endurance exercise performance. It has been called a waste product of anaerobic metabolism and has been believed to be responsible for the uncomfortable "burn" of intense exercise and directly responsible for the metabolic acidosis of exercise, leading to decreased muscle contractility and ultimately cessation of exercise. Although this premise has been commonly taught, it is not supported by the scientific literature and has led to a great deal of confusion among the sports medicine and exercise science communities. This review will provide the sports medicine clinician with an understanding of contemporary lactate theories, including lactate's role in energy production, its contributions to metabolic acidosis, and its function as an energy substrate for a variety of tissues. Lactate threshold concepts will also be discussed, including a practical approach to understanding prediction of performance and monitoring of training progress based on these parameters., (Copyright © 2016 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.)

This review summarizes the evidence base for using compression, massage, caloric replacement, cold, and heat as exercise recovery aids in sport.

Objective: To determine the amount of within-day variation of Concussion Symptom Severity Scores (CSSSs) in athletes with a clinically diagnosed concussion., Design: This prospective cohort study used a text-messaging robot to survey concussed athletes using the CSSS 5-times daily while they remained symptomatic., Setting: Academic sports concussion clinic at a large tertiary care medical center., Participants: Fourteen 14- to 22-year-old athletes with clinically diagnosed concussions were enrolled. All were injured in sport, still in season, English speaking, and used a cellular telephone to text message on a regular basis., Assessment of Risk Factors: This study did not collect any demographic or other information about the subjects. The only data collected were the individual responses to the questions in the Post Concussion Symptom Score (PCSS). The only aim of this study was to evaluate the variability in responses to the CSSS, which is calculated from the PCSS., Main Outcome Measurements: The primary outcome measure was a repeatability coefficient from a random-effects model. Concussion Symptom Severity Score, mean daily CSSS, and standard deviation of CSSS differences from the mean were also calculated., Results: Mean follow-up was 23.9 days. A total of 24 180 text messages were received from subjects. Eight hundred four complete surveys were analyzed. Repeatability coefficient was ±23.1., Conclusions: When surveyed frequently by a text-messaging robot, concussed athletes report poorly repeatable Sport Concussion Assessment Tool 2 (SCAT2) Symptom Severity Scores., Clinical Relevance: The SCAT2 Symptom Severity Score may be an invalid method of tracking concussion severity and resolution over time.

Introduction: The number of head injuries from skiing and snowboarding accidents is increasing among adolescents. Ski helmets reduce the risk of head injury. This study explored adolescent attitudes regarding helmet use., Methods: This pilot study included 11 high school students participating in a 1-hour focus group., Results: There was agreement that head injury is unlikely compared to other injuries, and use of helmets is determined by level of difficulty of the activity. Peer use makes personal use more acceptable and likely. Helmet cost is a minor barrier. Personal experience with a head injury increases use. Mandatory helmet use was viewed positively by most of the subjects., Conclusions: This pilot study suggests that, similar to bicycle helmet promotion programs, ski and snowboard helmet campaigns should focus on delivering a positive image of helmet use and peer acceptance.

Semianalytical (SA) ocean color models have advantages over conventional band ratio algorithms in that multiple ocean properties can be retrieved simultaneously from a single water-leaving radiance spectrum. However, the complexity of SA models has stalled their development, and operational implementation as optimal SA parameter values are hard to determine because of limitations in development data sets and the lack of robust tuning procedures. We present a procedure for optimizing SA ocean color models for global applications. The SA model to be optimized retrieves simultaneous estimates for chlorophyll (Chl) concentration, the absorption coefficient for dissolved and detrital materials [a(cdm)(443)], and the particulate backscatter coefficient [b(bp)(443)] from measurements of the normalized water-leaving radiance spectrum. Parameters for the model are tuned by simulated annealing as the global optimization protocol. We first evaluate the robustness of the tuning method using synthetic data sets, and we then apply the tuning procedure to an in situ data set. With the tuned SA parameters, the accuracy of retrievals found with the globally optimized model (the Garver-Siegel-Maritorena model version 1; hereafter GSM01) is excellent and results are comparable with the current Sea-viewing Wide Field-of-view sensor (SeaWiFS) algorithm for Chl. The advantage of the GSM01 model is that simultaneous retrievals of a(cdm)(443) and b(bp)(443) are made that greatly extend the nature of global applications that can be explored. Current limitations and further developments of the model are discussed.

Sensory potentials recorded from the dorsal cutaneous branch of the ulnar nerve (DCUN) help localize ulnar nerve lesions. This conduction technique was first described by Jabre in 1980. Abnormalities detected with this technique in control populations are commonly attributed to local trauma. Anatomic variations of cutaneous innervation to the dorsum of the hand, however, have been described. We recorded DCUN responses using both the normal DCUN stimulation point and a secondary site of stimulation at the radial aspect of the forearm in 44 patients. Eleven patients demonstrated a low-amplitude DCUN response. Four of the 11 patients demonstrated sensory potentials obtained from the routine recording site of the DCUN with radial stimulation. We conclude that an abnormal DCUN response may represent anomalous innervation. To avoid some errors in localization, radial forearm stimulation should be performed whenever the DCUN response is abnormal.

The synthesis of DNA has been studied by autoradiography and by measurements of tritiated thymidine ([3H]TdR) incorporation in cultured C3H/10T1/2 mouse embryo fibroblasts. The cells were first treated with 3-methylcholanthrene as an initiator and then with promoters according to schedules that produce oncogenic transformation. The levels of 3-methylcholanthrene used did not affect the growth or [3H]TdR incorporation of the cells. Treatment during the log phase of growth with 12-O-tetradecanoyl-phorbol-13-acetate, phorbol didecanoate, or 4alpha-phorbol didecanoate produced a transient inhibition of [3H]TdR incorporation with the maximum at 12 hr after treatment. This resulted in a temporary delay of growth followed by recovery of the normal cell-doubling time. Phorbol did not produce these effects, suggesting that the inhibition of DNA synthesis is associated with the process of promotion. Although treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate during stationary phase resulted in a 2- to 3-fold stimulation of [3H]TdR incorporation, multiple treatments spanning log and stationary phases were found to be necessary for promotion.

Morphological or oncogenic transformation of mouse embryo, C3H/10T1/2 Cl 8 fibroblasts was induced by methotrexate, 5-fluorouracil and 5-fluorodeoxyuridine. It is known that these compounds cause inhibition of thymidylate synthetase and, hence, depletion of deoxythymidine triphosphate (dTTP) and an increased ratio of deoxycytidine triphosphate (dCTP) to dTTP in the deoxyribonucleotide pools that are used for DNA synthesis in mammalian cells. This ratio is, in effect, increased by treating mammalian cells with arabinosyl cytosine and 5-azacytidine, which are converted into analogs of dCTP in mammalian cells and also induce oncogenic transformation of C3H/10T1/2 cells. By contrast, trifluorothymidine, 5-bromodeoxyuridine and 5-iododeoxyuridine, which are analogs of thymidine that in effect reduce the dCTP:dTTP ratio, did not induce oncogenic transformation. Moreover, thymidine was selectively lethal to tumorigenic C3H/10T1/2 cells and inhibited oncogenic transformation in cells treated with 5-fluorodeoxyuridine. These observations suggest that treatments that effectively increase the dCTP:dTTP ratio in mammalian cells facilitate oncogenic transformation of C3H/10T1/2 cells, whereas treatments that have the effect of decreasing this ratio inhibit transformation. However, dCyd did not induce oncogenic transformation of C3H/10T1/2 cells, although it has been shown to increase the dCTP:dTTP ratio in mammalian cells. Thus, increasing this ratio may not be sufficient to cause the transformation.

A 24-hr treatment of V79 Chinese hamster lung fibroblasts with 12.4 microM cholesterol 5 alpha, 6 alpha-epoxide induced 8-azaguanine-resistant mutants at frequencies 4.6- to 11.8-fold higher than the spontaneous mutation rate. We show that cholesterol epoxide, which is produced by in vivo cholesterol oxidation, is a weak direct-acting mutagen. Cholesterol epoxide was found to be accumulated by cells and transformed to cholestane-3 beta, 5 alpha, 6 beta-triol, which was more toxic and a more potent inhibitor of DNA synthesis than the epoxide but, at concentrations less than 17.8 microM, was not significantly mutagenic. Consideration of the rates of cholesterol epoxide conversion to cholestane triol shows that this conversion can result in abolition of the mutagenicity of the epoxide. Conditions under which conversion of the epoxide to the triol is low, as in the case of low epoxide hydrolase activity, favor mutagenicity whereas rapid conversion to triol favors cytotoxicity.

In medium containing concentrations of deoxycytidine that occur in vivo, 5-fluorodeoxyuridine induced mutation frequencies 6-90 fold greater than spontaneous mutant frequencies at two genetic loci in Chinese hamster cells. In medium lacking deoxycytidine, 5-fluorodeoxyuridine was more cytotoxic but induced no mutants. Hence, the effectiveness of cancer therapy with 5-fluorodeoxyuridine may be limited by self potentiated development of 5-fluorodeoxyuridine-resistant mutants and enhanced and prolonged by manipulating deoxycytidine metabolism.

Two-hr treatments with N-methyl- and N-ethyl-N'-nitro-N-nitrosoguanidines and ethyl methanesulfonate induced ouabain-resistant mutants in C3H/10T 1/2 cells. The alkylnitronitroso-guanidines gave linear dose-response curves and were more potent mutagens than were ethyl methanesulfonate and methyl methanesulfonate. These differences in potency were largely due to differences in the half-lives of the alkylating agents in culture medium. Differences in mutation frequencies at equitoxic concentrations of the alkylating agents are considered to reflect differences in the chemical mechanisms of alkylation and mutagenesis by the compounds. However, the frequencies of mutations produced at equitoxic concentrations were not uniformly associated with the nucleophilic selectivities of the compounds as expressed by their published Swain-Scott substrate constants. Whether or not followed by repeated replating, the yield of oncogenically transformed foci of asynchronous cells after treatment with the alkylating agents was so low that we could not obtain dose-response curves, and the yield may not be significant. By contrast, in previous experiments with N-methyl-N'-nitro-N-nitrosoguanidines and polycyclic hydrocarbons in Syrian hamster embryo fibroblasts and with ultraviolet light and polycyclic hydrocarbons in C3H/10T 1/2 cells, transformation occurred to an equal or greater extent than mutation measured in the same cells.

Low concentrations (less than or equal 20 mug/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 mug of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 mug/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; non-mutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH or US and low concentrations of AG were not mutants, but those from medium containing high concentrations (greater than or equal 30 mug/ml) of AG were mutants. Spontaneous and N-methyl-N-nitro-N-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (greater than or equal 30 mug/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH. HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.

Treatment of Chinese hamster V79 cells with dThd, dCyd, or dThd plus dCyd increased MNNG-induced AGr-, TGr-, and Ouar-mutant frequencies but did not significantly increase background mutant frequencies. All the AGr colonies that were isolated possessed phenotypes characteristic of HGPRT-deficient mutants, and the deoxyribonucleosides did not selectively affect the growth of the mutants, nor the selecting efficiency of AG, and did not significantly enhance background mutagenesis. These data show that both dThd and dCyd facilitated MNNG-induced mutagenesis. This facilitation was maximal when cells were exposed to the deoxyribonucleosides throughout the first doubling time (24 h) after treatment with MNNG and for 4 more doubling times prior to mutant selection with AG. This indicates that one round of DNA replication was sufficient for mispairing of methylated bases in the DNA with the C and T provided by the deoxyribonucleosides, and that 4-6 doublings prior to mutant selection with AG were necessary to deplete pre-existing hypoxanthine: guanine phosphoribosyl transferase in newly mutated cells. The dCyd facilitated mutagenesis by FdUrd, which was not mutagenic without dCyd, indicating that increased dCTP:dTTP ratios were mutagenic. Treatment with FdUrd plus dCyd also induced FdUrdr cells, suggesting that inhibition of dCyd utilization may prevent the development of FdUrd-resistance in cancer chemotherapy. Although dCyd and dThd facilitated mutagenesis in cells treated with monofunctional alkylating agents that methylate DNA oxygens, facilitation of mutagenesis did not occur in cells treated with BCNU, which cross links DNA, nor with benzo(a)pyrene and aflatoxin B1, which are frame shift mutagens, nor with MMS, which produces barely detectable levels of O-methylation in DNA. Virtually non toxic concentrations of dThd potentiated the cytotoxicity of MNNG more than 10-fold but that of MMS was potentiated only about 2-fold showing that O-alkylation of DNA was associated not only with the facilitation of mutagenesis but also with the potentiation of cytotoxicity. The potentiation of MNNG-induced cytotoxicity was maximal in V79 and L1210 cells after only 2 h treatment with dThd, showing that not even one round of DNA replication was necessary for this potentiation. Moreover, dCyd abolished the potentiation, and, at equitoxic concentrations, MNNG induced higher mutant frequencies than did MMS. These data show that the mechanisms by which methylating agents plus dThd induce mutagenesis are fundamentally different from their mechanisms of cytotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholesterol, cholesterol-5 alpha, 6 alpha-epoxide, cholesterol-5 beta, 6 beta-epoxide and cholestane-3 beta,5 alpha,6 beta-triol were tested for their ability to induce mutations at the Na+/K+-ATPase loci of the Chinese hamster V79 cells. None of these compounds induced ouabain-resistant mutations compared to the background mutation frequency in the control cells. These compounds were further tested for their ability to inhibit intercellular communication, using the Chinese hamster V79 cell metabolic cooperation assay. The diastereomeric epoxides and cholestane-triol, but not cholesterol, were found to be inhibitors of intercellular communication in a manner similar to other known tumor promoters.

The oxidation of cholesterol proceeds as part of the lipid peroxidation process in membranes. Several oxidation products characteristic of a free-radical mechanism are formed and some can serve as indices of the nature and extent of cholesterol oxidation and of lipid peroxidation in general. Among the most typical oxidation products of lipid peroxide-dependent propagation reactions are the enantiomeric 5,6-epoxides and 7-ketocholestanol. Small amounts of these compounds may persist in tissues experiencing lipid peroxidation at a low but steady flux of free-radical reactions. Supporting evidence includes the routine detection of small quantities of cholesterol epoxides in tissues of normal animals, and the increase of these epoxides under conditions of oxidant stress or antioxidant deficiency. Conversion of cholesterol epoxides to cholestane triol is expected in cells possessing cholesterol epoxide hydrolase. All of these oxidation products possess remarkable cytotoxicity (at least part of which may be due to effects on the cell membrane) causing an increase in intracellular calcium. The cholesterol epoxides are also weakly mutagenic, although the mechanism for this mutagenicity remains to be clarified. In contrast, the other lipid epoxides normally encountered in tissues (chiefly fatty acid epoxides) are not mutagenic, and are much less toxic than the oxysterols described. The cytotoxicity of several oxysterols may be due to a number of mechanisms. That only the epoxides are mutagenic suggests that genotoxicity is a function of their electrophilic reactivity. This is not consistently apparent with the other compounds examined.

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.

A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol.

The uptake, metabolism and alkylating properties of the diastereomeric cholesterol epoxides were studied using Chinese hamster lung fibroblasts (V79 cells). Specific emphasis is given to the comparative cyto- and geno-toxic effects of cholesterol 5 beta,6 beta-epoxide (beta CE) and cholesterol 5 alpha,6 alpha-epoxide (alpha CE) and data are provided for the first time indicating that beta CE can induce more 6-thioguanine-resistant cells than alpha CE. Cholesterol 5 beta,6 beta-epoxide induced colonies of cells resistant to 6-thioguanine at 2-3-fold the frequencies observed with the alpha-isomer, but neither compound produced ouabain-resistant colonies. The cytotoxicity (LD50) of alpha CE was estimated to be 45-50 microM whereas beta CE displayed an LD50 of 25-29 microM. Inhibition of DNA synthesis (IC50) was observed over the same dose ranges as the LD50 for each epoxide isomer. The epoxides were assimilated by cells to an equal extent, however, beta CE was metabolized to cholestane 3 beta,5 alpha-6 beta-triol twice as rapidly as the alpha-isomer. Both epoxides reacted with 4-(4'-nitrobenzyl)-pyridine to a similar extent, and with identical nucleophilic selectivity at pH 7.4, but their alkylating activity was estimated on this basis to be two orders of magnitude less than methyl methanesulfonate. Binding experiments with the DNA or cultured V79 cells or with calf-thymus DNA indicated that interactions were noncovalent and DNA binding did not correlate with the potency of the epoxides to induce the 6-thioguanine-resistant phenotype. Our results could be interpreted as indicating that both cholesterol epoxide isomers are weak mutagens or that they might induce some epigenetic event repressing the hypoxanthine guanine-phosphoribosyltransferase gene. The similarity of the epoxides' alkylating activity and their DNA-binding properties are inconsistent with their different potencies in inducing the 6-thioguanine-resistant phenotype, suggesting that the mechanism leading to this phenotype is not necessarily the result of DNA alkylation.

Hypoxanthine (Hx), thymidine (TdR) and deoxycytidine (CdR), at concentrations of 10(-5) M increased the yield of 8-azaguanine-resistant (AzGr) mutants induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster V79 cells. The cytotoxicity of MNNG was increased 2-fold in the presence of Hx, and more than 10-fold in the presence of TdR. This cytotoxic effect of TdR was abolished by equal concentrations of CdR, which by itself did not increase the cytotoxicity of MNNG. However, the yield of MNNG-induced AzGr colonies was increased 2--10-fold in the presence of both CdR and TdR. The AzGr colonies displayed phenotypes characteristic of hypoxanthine: guaninephosphoribosyltransferase-deficient (HGPRT-) mutants, or indicative of mutant HGPRT with altered substrate affinities. The nucleosides did not affect the growth or expression time of the HGPRT- mutants; the same extent of alkali-labile DNA damage occurred in cells treated with alkylating agents in the presence and absence of TdR and CdR; and the increase in mutation frequency in the presence of these nucleosides occurred not only with MNNG, but also with ethylating agents. Nucleosides treated with MNNG were not mutagenic, and treatment of the cells with TdR and CdR only prior to treatment with MNNG or only during selection with AzG did not increase the induced mutation frequency. Therefore, the interpretation is proposed that CdR, TdR and Hx produce nucleotide-pool imbalances that increase lethal and mutagenic errors of replication of alkylated DNA.