Carbon Footprint of Minor Foot and Ankle Surgery: A Randomized Controlled Trial.

Background: Climate change poses a substantial threat to human health, and operating rooms (ORs) have an outsized environmental impact. The Program for Research in Sustainable Medicine (PRiSM) designed a protocol for minor foot and ankle surgery intended to reduce waste, streamline instrument trays, and minimize laundry. We conducted a randomized controlled trial to compare the carbon footprint of procedures performed using the PRiSM protocol vs a traditional protocol. Methods: Forty adult patients undergoing foreign body removal, hammertoe correction, toe amputation, hardware removal, mass excision, or gastrocnemius recession were randomized to the PRiSM or our "Traditional" protocol. The PRiSM protocol used a smaller instrument tray, fewer drapes and towels, and minimal positioning blankets. No changes were made to surgical site preparation or operative techniques. Environmental impact was estimated using the carbon footprint, measured in kilograms of carbon dioxide equivalents (CO2e). Emissions associated with OR waste, instrument processing, and laundry were calculated. Results: On average, PRiSM cases had a smaller carbon footprint than Traditional cases (17.3 kg CO2e [SD = 3.2] vs 20.6 kg CO2e [SD = 2.0], P < .001). Waste-associated emissions from PRiSM cases were reduced (16.0 kg CO2e [SD = 2.7] vs 18.4 kg CO2e [SD = 1.8], P = .002), as were modeled instrument processing-related emissions (0.34 vs 0.91 kg CO2e). One superficial surgical site infection occurred in each group. Conclusion: We found a small but statistically significant reduction in the environmental impact of minor foot and ankle surgery when using the PRiSM vs Traditional protocol. The environmental impact of these cases was dominated by plastic waste-related emissions. Orthopaedic surgeons should think critically about what components of their surgical setup are truly necessary for patient care, as minor changes in product utilization can have significant impacts on waste and greenhouse gas emissions. Level of Evidence: Level I, randomized controlled trial.

Primary Care Physician Preferences Regarding Communication from Orthopaedic Surgeons.

BACKGROUND: Musculoskeletal consultations constitute a growing portion of primary care physician (PCP) referrals. Optimizing communication between PCPs and orthopaedists can potentially reduce time spent in the electronic medical record (EMR) as well as physician burnout. Little is known about the preferences of PCPs regarding communication from orthopaedic surgeons. Hence, the present study investigated, across a large health network, the preferences of PCPs regarding communication from orthopaedists. METHODS: A total of 175 PCPs across 15 practices within our health network were surveyed. These providers universally utilized Epic as their EMR platform. Five-point, labeled Likert scales were utilized to assess the PCP-perceived importance of communication from orthopaedists in specific clinical scenarios. PCPs were further asked to report their preferred method of communication in each scenario and their overall interest in communication from orthopaedists. Logistic regression analyses were performed to determine whether any PCP characteristics were associated with the preferred method of communication and the overall PCP interest in communication from orthopaedists. RESULTS: A total of 107 PCPs (61.1%) responded to the survey. PCPs most commonly rated communication from orthopaedists as highly important in the scenario of an orthopaedist needing information from the PCP. In this scenario, PCPs preferred to receive an Epic Staff Message. Scenarios involving a recommendation for surgery, hospitalization, or a major clinical change were also rated as highly important. In these scenarios, an Epic CC'd Chart rather than a Staff Message was preferred. Increased after-hours EMR use was associated with diminished odds of having a high interest in communication from orthopaedists (odds ratio, 0.65; 95% confidence interval, 0.48 to 0.88; p = 0.005). Ninety-three PCPs (86.9%) reported spending 1 to 1.5 hours or more per day in Epic after normal clinical hours, and 27 (25.2%) spent >3 hours per day. Forty-six PCPs (43.0%) reported experiencing ≥1 symptom of burnout. CONCLUSIONS: There were distinct preferences among PCPs regarding clinical communication from orthopaedic surgeons. There was also evidence of substantial burnout and after-hours work effort by PCPs. These results may help to optimize communication between PCPs and orthopaedists while reducing the amount of time that PCPs spend in the EMR.

Non-mutagenic impurities - Recent industry experience of using dose durational limits in drug development.

Absence of clear guidance on the qualification threshold for non-mutagenic impurities during clinical development is a source of inconsistency in both sponsor qualification approaches and health authority requests. A survey was conducted in March 2020 with 6 member companies of the European Federation of Pharmaceutical Industries and Associations (EFPIA). Thirteen examples were gathered of where non-International Council for Harmonisation (ICH) limits have been used in regulatory submissions for various indications and stages of development, together with the regulatory outcomes. As expected, few challenges were faced in early clinical development, with health authorities generally commenting that sponsors should work towards ICH Q3A and Q3B guideline specification limits as development progresses. However, inconsistent health authority requests were noted even for early phase clinical trials in late-stage oncology patients. For an optimised use of resources, consistent approaches would have the benefit of supporting faster access of safe medicines to patients while including Replacement, Reduction and Refinement (the 3Rs) considerations with respect to animal testing.

Efficiency and perceived safety of foot and ankle procedures performed on the preoperative stretcher versus operating room table.

BACKGROUND: Foot and ankle surgeons often perform minor surgeries on the preoperative stretcher instead of the operating room table. We examined whether stretcher-based and operating room table-based procedures differed with respect to operating room efficiency and staff perceptions. METHODS: We retrospectively reviewed medical records of patients undergoing minor foot and ankle surgery at an ambulatory surgery centre. We collected 'time to start', the duration between patient arrival in the operating room and incision time, and 'time to exit', the duration between procedure end time and patient exit from the operating room. Staff were surveyed regarding their perceptions of stretcher-based and operating room table-based procedures. RESULTS: 'Time to start' was significantly shorter for stretcher-based procedures, but 'time to exit' was not. Seventeen (81%) staff members thought stretcher-based procedures increased operating room efficiency. Thirteen (62%) thought stretcher-based procedures bettered staff safety. Nineteen (91%) thought stretcher-based procedures were equivalent to or better than operating room table-based procedures for patient safety. Most (67%) would recommend stretcher-based procedures. CONCLUSION: We found small but significant time savings associated with stretcher-based procedures. Without adapting surgical scheduling practices, the impact of stretcher-based procedures on overall operating room efficiency is questionable. Nevertheless, the majority of OR staff think stretcher-based procedures increase OR efficiency and are safer for staff. LEVEL OF EVIDENCE: Level IV, Retrospective case series.

The Effects of Sociodemographic Factors on Baseline Patient-Reported Outcome Measures in Patients with Foot and Ankle Conditions.

BACKGROUND: Racial and ethnic care disparities persist within orthopaedics in the United States. This study aimed to deepen our understanding of which sociodemographic factors most impact patient-reported outcome measure (PROM) score variation and may explain racial and ethnic disparities in PROM scores. METHODS: We retrospectively reviewed baseline PROMIS (Patient-Reported Outcomes Measurement Information System) Global-Physical (PGP) and PROMIS Global-Mental (PGM) scores of 23,171 foot and ankle patients who completed the instrument from 2016 to 2021. A series of regression models was used to evaluate scores by race and ethnicity after adjusting in a stepwise fashion for household income, education level, primary language, Charlson Comorbidity Index (CCI), sex, and age. Full models were utilized to compare independent effects of predictors. RESULTS: For the PGP and PGM, adjusting for income, education level, and CCI reduced racial disparity by 61% and 54%, respectively, and adjusting for education level, language, and income reduced ethnic disparity by 67% and 65%, respectively. Full models revealed that an education level of high school or less and a severe CCI had the largest negative effects on scores. CONCLUSIONS: Education level, primary language, income, and CCI explained the majority (but not all) of the racial and ethnic disparities in our cohort. Among the explored factors, education level and CCI were predominant drivers of PROM score variation. LEVEL OF EVIDENCE: Prognostic Level IV . See Instructions for Authors for a complete description of levels of evidence.

Scope of the Problem: Ankle Arthroscopy.

Ankle arthroscopy has seen increased utilization and application in recent years. Through the advent of improved instrumentation and techniques, indications have been expanded to include the management of traumatic, degenerative, inflammatory, and neoplastic conditions. It is important to review anterior and posterior ankle arthroscopies along with the history, pertinent anatomy, techniques, indications, and complications as well as gain insight into the future of ankle arthroscopy.

Scope of the Problem: Tendoscopy and Small Joint Arthroscopy of the Foot and Ankle.

It is important to identify and describe practical applications of arthroscopy in the management of foot and ankle pathology. Utilization of the arthroscope provides a minimally invasive means of evaluating and addressing pathology. It obviates the need for a large open approach, which has additional value in the setting of a multiprocedure surgery. In addition to reducing surgical time, arthroscopy provides a potentially enhanced field of view and an adequate working space to address injury. As interest in minimally invasive options grows, the need for safe, effective tendoscopic and arthroscopic options in the foot and ankle increases. A clear and high-yield reference is needed with which to approach these procedures.

The Canonical Wnt Signaling Pathway Inhibits the Glucocorticoid Receptor Signaling Pathway in the Trabecular Meshwork.

Glucocorticoid-induced glaucoma is a secondary open-angle glaucoma. About 40% of the general population may develop elevated intraocular pressure on prolonged glucocorticoid treatment secondary to damages in the trabecular meshwork (TM), a tissue that regulates intraocular pressure. Therefore, identifying the key molecules responsible for glucocorticoid-induced ocular hypertension is crucial. In this study, Dickkopf-related protein 1 (Dkk1), a canonical Wnt signaling inhibitor, was found to be elevated in the aqueous humor and TM of glaucoma patients. At the signaling level, Dkk1 enhanced glucocorticoid receptor (GR) signaling, whereas Dkk1 knockdown or Wnt signaling activators decreased GR signaling in human TM cells as indicated by luciferase assays. Similarly, activation of the GR signaling inhibited Wnt signaling. At the protein level, glucocorticoid-induced extracellular matrix was inhibited by Wnt activation using Wnt activators or Dkk1 knockdown in primary human TM cells. In contrast, inhibition of canonical Wnt signaling by ß-catenin knockdown increased glucocorticoid-induced extracellular matrix proteins. At the physiological level, adenovirus-mediated Wnt3a expression decreased glucocorticoid-induced ocular hypertension in mouse eyes. In summary, Wnt and GR signaling inhibit each other in the TM, and canonical Wnt signaling activators may prevent the adverse effect of glucocorticoids in the eye.

Pathologic Hip Fracture by Virtue of a Rare Osseous Manifestation of Gout: A Case Report.

CASE: A 76-year-old woman without a personal or family history of gout presented with complaints of left hip pain after a mechanical fall from her wheelchair. Advanced imaging revealed a nonspecific lesion and nondisplaced fracture of the femoral neck. Intraoperative biopsy from the lesion/fracture demonstrated tophaceous gout. CONCLUSION: Fractures resulting from osseous manifestations of the gout are rare with this report describing a hip fracture secondary to tophaceous gout. We emphasize the importance of including this potential etiology in the differential diagnosis of elderly patients presenting with hip pain, with or without a known history of gout.

Effects of a monocarboxylate transport 1 inhibitor, AZD3965, on retinal and visual function in the rat.

BACKGROUND AND PURPOSE: Inhibition of monocarboxylate transport 1 (MCT1) is of interest in targeting highly glycolytic tumours. However, MCT1 is expressed in retina, and so inhibition of MCT1 could affect retinal function. EXPERIMENTAL APPROACH: AZD3965, an MCT1 inhibitor selected for clinical development, and two additional MCT1 inhibitors were evaluated for effects on visual acuity in albino (Han Wistar) rats. The effects of AZD3965 on visual acuity and electroretinography (ERG) were further investigated in pigmented (Long-Evans) rats, with dosing for up to 7 days. KEY RESULTS: All three MCT1 inhibitors reduced visual acuity within 2 h of dosing, suggesting a class effect. The deficit caused by AZD3965 (1,000 mg·kg-1 p.o. per day for 4 days) in Long Evans rats recovered to pre-dose levels 7 days after cessation of dosing. AZD3965 (50 to 1,000 mg·kg-1 p.o.) reduced the amplitude of scotopic a- and b-waves, and photopic b-wave of the ERG in a dose-related fashion, within 2 h of dosing. The effects on the scotopic ERG had diminished by Day 7 of dosing, demonstrating partial restoration of function despite continued treatment. Seven days after cessation of dosing at the highest dose tested (1,000 mg·kg-1 ), there was recovery of both scotopic a- and b- waves and, to a lesser extent, photopic b-wave. ERG was affected at lower plasma exposures than was visual function. CONCLUSIONS AND IMPLICATIONS: This study clarifies the role of the MCT1 transporter in retinal function. The monitorability of the functional effects on the retina enabled safe clinical use of AZD3965.

Quality and Utility of Immediate Formal Postoperative Radiographs in Ankle Fractures.

BACKGROUND: Patients who undergo internal fixation of ankle fractures commonly have postoperative imaging performed immediately after surgery. As these patients typically are typically immobilized, radiographs provide limited visualization. The purpose of this study was to evaluate the utility and quality of formal radiographs performed immediately following ankle fracture surgery. METHODS: Ankle fractures undergoing open reduction and internal fixation at a single institution from January 1, 2011, to January 1, 2013, were reviewed. Intraoperative and formal postoperative radiographs were evaluated using defined parameters. The postoperative images were compared with the intraoperative fluoroscopic images in terms of quality. Postoperative complications were evaluated in terms of fracture displacement, hardware malpositioning, and need for return to the operating room. A total of 411 patients with 413 ankle fractures underwent surgical fixation, with 271 patients undergoing formal postoperative radiographs. RESULTS: Twenty-eight patients (10.3%) had 3 good quality postoperative views of the ankle, with the lateral (35.2%) and mortise (41.3%) views least commonly performed with good technique. None of the patients without radiographs had a complication that could have been detected earlier using postoperative radiographs. No patients required return to the operating room based on immediate postoperative radiographs. Postoperative radiographs cost $191 per patient. CONCLUSION: The routine use of formal postoperative radiographs prior to discharge from the hospital did not provide additional value to the patient or orthopedic surgeon. The quality of these images was generally inferior to the quality of those obtained and saved intraoperatively due to malrotation and overlying cast material. To reduce cost and radiation exposure, formal postoperative radiographs should be obtained only in specific circumstances, such as increasing postoperative pain, marginal fixation, or instability. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Circumventing seizure activity in a series of G protein coupled receptor 119 (GPR119) agonists.

Agonism of GPR119 is viewed as a potential therapeutic approach for the treatment of type II diabetes and other elements of metabolic syndrome. During progression of a previously disclosed candidate 1 through mice toxicity studies, we observed tonic-clonic convulsions in several mice at high doses. An in vitro hippocampal brain slice assay was used to assess the seizure liability of subsequent compounds, leading to the identification of an aryl sulfone as a replacement for the 3-cyano pyridyl group. Subsequent optimization to improve the overall profile, specifically with regard to hERG activity, led to alkyl sulfone 16. This compound did not cause tonic-clonic convulsions in mice, had a good pharmacokinetic profile, and displayed in vivo efficacy in murine models. Importantly, it was shown to be effective in wild-type (WT) but not GPR119 knockout (KO) animals, consistent with the pharmacology observed being due to agonism of GPR119.

Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca(2+) concentration in rat carotid body glomus cells.

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca(2+) concentration ([Ca(2+)]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K(+) channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1mM AICAR and 0.1-0.5mM A769662) did not inhibit TASK activity or cause depolarization during acute (10min) or prolonged (2-3h) exposure. Hypoxia inhibited TASK activity by ∼70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15-40min) failed to increase [Ca(2+)]i in glomus cells. Compound C (40µM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca(2+)] in isolated rat carotid body glomus cells.

An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride.

The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.

Polyphenol-rich cranberry juice has a neutral effect on endothelial function but decreases the fraction of osteocalcin-expressing endothelial progenitor cells.

PURPOSE: Cranberry juice (CJ) contains a remarkably high concentration of polyphenols, considered to be beneficial for cardiovascular and bone health. The current double-blind, randomized study was designed to test whether daily consumption of double-strength Ocean Spray light CJ (2 × 230 ml) over 4 months has beneficial effects on vascular function and on endothelial progenitor cells (EPCs) carrying the osteoblastic marker osteocalcin in particular. METHODS: A total of 84 participants (49.5 ± 16.2 years) with peripheral endothelial dysfunction and cardiovascular risk factors were enrolled in this double-blind, randomized, controlled trial (69 completed the 4-month protocol-32 in the CJ group and 37 in the placebo group, respectively). Vascular responses to reactive hyperemia were measured non-invasively by peripheral arterial tonometry (EndoPAT). Peripheral blood mononuclear cells were stained for EPC markers, as well as osteocalcin, and counted by flow cytometry. RESULTS: Baseline characteristics were similar in both groups. The effect of CJ on peripheral endothelial function and on circulating EPC counts (CD34(+)/CD133(+)/KDR(+)) did not change during the study. A high percentage of EPCs expressed osteocalcin (59.4 ± 35.7%). CJ, as compared to placebo, induced a decrease in the fraction of EPCs expressing osteocalcin (-8.64 ± 48.98 and 19.13 ± 46.11%, respectively, p = 0.019). Systemic levels of the adhesion marker ICAM correlated significantly with the number of EPCs expressing osteocalcin. CONCLUSIONS: The study demonstrated that long-term supplementation of polyphenol-rich CJ did not improve peripheral endothelial function. However, the decrease in the fraction of osteocalcin+ EPCs suggests a potential beneficial effect of polyphenol-rich CJ.

Tadalafil alleviates muscle ischemia in patients with Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is a progressive X-linked muscle wasting disease for which there is no treatment. Like Duchenne muscular dystrophy (DMD), BMD is caused by mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the muscle sarcolemma. Among these is neuronal nitric oxide synthase (nNOSµ), which requires certain spectrin-like repeats in dystrophin's rod domain and the adaptor protein α-syntrophin to be targeted to the sarcolemma. When healthy skeletal muscle is subjected to exercise, sarcolemmal nNOSµ-derived NO attenuates local α-adrenergic vasoconstriction, thereby optimizing perfusion of muscle. We found previously that this protective mechanism is defective-causing functional muscle ischemia-in dystrophin-deficient muscles of the mdx mouse (a model of DMD) and of children with DMD, in whom nNOSµ is mislocalized to the cytosol instead of the sarcolemma. We report that this protective mechanism also is defective in men with BMD in whom the most common dystrophin mutations disrupt sarcolemmal targeting of nNOSµ. In these men, the vasoconstrictor response, measured as a decrease in muscle oxygenation, to reflex sympathetic activation is not appropriately attenuated during exercise of the dystrophic muscles. In a randomized placebo-controlled crossover trial, we show that functional muscle ischemia is alleviated and normal blood flow regulation is fully restored in the muscles of men with BMD by boosting NO-cGMP (guanosine 3',5'-monophosphate) signaling with a single dose of the drug tadalafil, a phosphodiesterase 5A inhibitor. These results further support an essential role for sarcolemmal nNOSµ in the normal modulation of sympathetic vasoconstriction in exercising human skeletal muscle and implicate the NO-cGMP pathway as a putative new target for treating BMD.

Modulation of K2P3.1 (TASK-1), K2P9.1 (TASK-3), and TASK-1/3 heteromer by reactive oxygen species.

Reactive oxygen species (ROS) generated by mitochondria or NADPH oxidase have been implicated in the inhibition of K(+) current by hypoxia in chemoreceptor cells. As TASKs are highly active background K(+) channels in these cells, we studied the role of ROS in hypoxia-induced inhibition of TASKs. In HeLa cells expressing TASKs, H(2)O(2) applied to inside-out patches activated TASK-1, TASK-3, and TASK-1/3 heteromer starting at ~16 mM. When applied to cell-attached or outside-out patches, 326 mM H(2)O(2) did not affect TASK activity. Other K(2P) channels (TREK-1, TREK-2, TASK-2, TALK-1, TRESK) were not affected by H(2)O(2) (tested up to 326 mM). A reducing agent (dithiothreitol) and a cysteine-modifying agent (2-aminoethyl methanethiosulfonate hydrobromide) had no effect on basal TASK activity and did not block the H(2)O(2)-induced increase in channel activity. A TASK mutant in which the C-terminus of TASK-3 was replaced with that of TREK-2 showed a normal sensitivity to H(2)O(2). Xanthine/xanthine oxidase mixture used to generate superoxide radical showed no effect on TASK-1, TASK-3, and TASK-1/3 heteromer from either side of the membrane, but it strongly activated TASK-2 from the extracellular side. Acute H(2)O(2) (32-326 mM) exposure did not affect hSlo1/b1(BK) expressed in HeLa cells and BK in carotid body glomus cells. In carotid body glomus cells, adrenal cortical cells, and cerebellar granule neurons that show abundant hypoxia-sensitive TASK activity, H(2)O(2) (>16 mM) activated the channels only when applied intracellularly, similar to that observed with cloned TASKs. These findings show that ROS do not support or inhibit TASK and BK activity and therefore are unlikely to be the hypoxic signal that causes cell excitation via inhibition of these K(+) channels.

Agonists at the serotonin receptor (5-HT(1A)) protect the retina from severe photo-oxidative stress.

PURPOSE: 5-HT(1A) agonists are neuroprotective in CNS injury models. The authors evaluated the efficacy of 5-HT(1A) agonists to protect the retina from severe blue light-induced photo-oxidative damage. METHODS: Albino rats were dosed (subcutaneously) with AL-8309A, 8-OH DPAT, or buspirone once or three times before 6-hour exposure to blue light. Electroretinograms (ERGs) were measured to assess retinal function, and retinal damage was evaluated by light microscopy. Topical ocular dosing with 1.75% AL-8309B was also evaluated. Rats were dosed with WAY-100635, a 5-HT(1A) antagonist, to determine whether protection required activation of the 5-HT(1A) receptor. RESULTS: ERG response amplitudes were significantly (P < 0.05) depressed more than 66% in vehicle-dosed rats after light exposure. ERGs were significantly higher in rats treated with AL-8309A (0.1-30 mg/kg), 8-OH DPAT (0.1-1 mg/kg), buspirone (5-20 mg/kg) or topical ocular with 1.75% AL-8309B. Retinas from AL-8309A and 8-OH DPAT-treated rats were devoid of histologic lesions. Significant protection was measured in rats dosed once 0, 24, or 48 hours before light exposure. Protection provided by dosing with AL-8309B or 8-OH DPAT was inhibited in rats predosed with WAY-100635. CONCLUSIONS: 5-HT(1A) agonists provided potent and complete functional and structural protection. Protection was inhibited by treatment with WAY-100635, confirming the requirement for activating the 5-HT(1A) receptor in initiating this survival pathway. Single-dose experiments with AL-8309A suggest that the mechanism of protection is rapidly activated and protection persists for 48 hours. AL-8309B (1.75%) was effective after topical ocular dosing. AL-8309B is under evaluation in the clinic and may be useful in treating age-related macular degeneration.

Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage.

The formation of deoxyribonucleic acid (DNA) adducts can have important and adverse consequences for cellular and whole organism function. Available methods for identification of DNA damage and quantification of adducts are reviewed. Analyses can be performed on various samples including tissues, isolated cells, and intact or hydrolyzed (digested) DNA from a variety of biological samples of interest for monitoring in humans. Sensitivity and specificity are considered key factors for selecting the type of method for assessing DNA perturbation. The amount of DNA needed for analysis is dependent upon the method and ranges widely, from <1 microg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled ((14)C- and (3)H-) binding, (32)P-postlabeling, and methods dependent on gas chromatography (GC) or high-performance liquid chromatography (HPLC) with detection by electron capture, electrochemical detection, single or tandem mass spectrometry, or accelerator mass spectrometry. Sensitivity is ranked, and ranges from approximately 1 adduct in 10(4) to 10(12) nucleotides. A brief overview of oxidatively generated DNA damage is also presented. Assay limitations are discussed along with issues that may have impact on the reliability of results, such as sample collection, processing, and storage. Although certain methodologies are mature, improving technology will continue to enhance the specificity and sensitivity of adduct analysis. Because limited guidance and recommendations exist for adduct analysis, this effort supports the HESI Committee goal of developing a framework for use of DNA adduct data in risk assessment.

Recommendations for design of the rat comet assay.

Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25-100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.