Watching a signaling protein function: What has been learned over four decades of time-resolved studies of photoactive yellow protein.

Photoactive yellow protein (PYP) is a signaling protein whose internal p-coumaric acid chromophore undergoes reversible, light-induced trans-to-cis isomerization, which triggers a sequence of structural changes that ultimately lead to a signaling state. Since its discovery nearly 40 years ago, PYP has attracted much interest and has become one of the most extensively studied proteins found in nature. The method of time-resolved crystallography, pioneered by Keith Moffat, has successfully characterized intermediates in the PYP photocycle at near atomic resolution over 12 decades of time down to the sub-picosecond time scale, allowing one to stitch together a movie and literally watch a protein as it functions. But how close to reality is this movie? To address this question, results from numerous complementary time-resolved techniques including x-ray crystallography, x-ray scattering, and spectroscopy are discussed. Emerging from spectroscopic studies is a general consensus that three time constants are required to model the excited state relaxation, with a highly strained ground-state cis intermediate formed in less than 2.4 ps. Persistent strain drives the sequence of structural transitions that ultimately produce the signaling state. Crystal packing forces produce a restoring force that slows somewhat the rates of interconversion between the intermediates. Moreover, the solvent composition surrounding PYP can influence the number and structures of intermediates as well as the rates at which they interconvert. When chloride is present, the PYP photocycle in a crystal closely tracks that in solution, which suggests the epic movie of the PYP photocycle is indeed based in reality.

Assessment of Cimetropium Bromide Use for the Detection of Gastric Neoplasms During Esophagogastroduodenoscopy.

Importance: Esophagogastroduodenoscopy (EGD) is a common procedure used to examine upper gastrointestinal diseases. Although cimetropium bromide and other antispasmodic agents are commonly administered as premedication to inhibit peristalsis during EGD examination, there are few data regarding the benefits of cimetropium bromide for the detection of gastric neoplasms. Objective: To investigate the association between the use of cimetropium bromide as premedication and gastric neoplasm detection rates during EGD examination. Design, Setting, and Participants: This propensity score-matched retrospective cohort study included 67 683 participants who received EGD screening at the Health Promotion Center of Seoul St. Mary's Hospital, The Catholic University of Korea, from January 2, 2010, to June 30, 2017. Data were analyzed from April 1 to December 30, 2021. Exposures: Participants were divided into 2 groups: those who received cimetropium bromide before EGD examination (intervention group) and those who did not (control group). Main Outcomes and Measures: Gastric neoplasm detection rates. Results: Among 67 683 participants, the mean (SD) age was 48.6 (10.8) years, and 36 517 participants (54.0%) were male; all participants were Asian (a racially homogenous population). Of those, 28 280 participants (41.8%; mean [SD] age, 50.3 [10.6] years; 57.8% male) received cimetropium bromide, and 39 403 participants (58.2%; mean [SD] age, 47.4 [10.8] years; 51.2% male) did not. Propensity score matching based on confounding variables yielded 41 670 matched participants (20 835 pairs). Detected lesions included 52 dysplasias (0.12%), 40 early cancers (0.10%), 7 advanced cancers (0.02%), and 3 lymphomas (0.01%). Gastric neoplasm detection rates were significantly higher in the intervention group (63 participants [0.30%]) vs the control group (39 participants [0.19%]; P = .02). A significant difference in the combined detection rate of dysplasia and early gastric cancer was found between those in the intervention group (57 participants [0.27%]) vs the control group (35 participants [0.17%]; P = .02). For small gastric lesions (<1 cm), those who received cimetropium bromide had higher detection rates (24 participants [0.12%]) than those who did not (11 participants [0.05%]; P = .03). Lesions in the gastric body were detected significantly more often in the intervention group (34 participants [0.16%]) vs the control group (15 participants [0.07%]; P = .007). In multivariate analyses involving all 67 683 participants, the use of cimetropium bromide was more likely to detect gastric neoplasms compared with nonuse (odds ratio, 1.42; 95% CI, 1.04-1.95; P = .03). Conclusions and Relevance: In this study, the use of cimetropium bromide as premedication was significantly associated with increased gastric neoplasm detection rates during EGD screening, and lesions in the gastric body were detected more frequently among those who received cimetropium bromide compared with those who did not. These findings suggest that cimetropium bromide may be considered as premedication before EGD examination among individuals with no contraindications.

Time-resolved X-ray scattering studies of proteins.

Time-resolved small- and wide-angle X-ray scattering studies of proteins in solution based on the pump-probe approach unveil structural information from intermediates over a broad range of length and time scales. In spite of the promise of this methodology, only a fraction of the wealth of information encoded in scattering data has been extracted in studies performed thus far. Here, we discuss the methodology, summarize results from recent time-resolved X-ray scattering studies, and examine the potential to extract additional information from these scattering curves.

Implementation effect of institutional policy of EGD observation time on neoplasm detection.

BACKGROUND AND AIMS: The observation time in EGD is associated with detection rate of premalignant or neoplastic lesions in the upper GI (UGI) tract. The aim of this study was to evaluate an institutional policy of EGD observation time on the detection rate of UGI neoplasms. METHODS: From July 2017 to March 2019, all endoscopists were requested to comply with our institutional policy of spending more than 3 minutes of observation time in every screening EGD. Observation time was defined as the time from when the endoscope reached the duodenum to when it was withdrawn. We obtained a neoplasm detection rate (NDR) during this period and compared it with that of a baseline period from 2009 to 2015. RESULTS: During the study period, 30,506 EGDs were performed. Mean subject age was 49.9 ± 10.5 years, and 56.5% were men. All endoscopists achieved an average EGD observation time of more than 3 minutes during this period. Mean observation time was 3:35 ± 0:50, which was significantly longer than the baseline (2:38 ± 0:21, P < .001). NDR was .33%, which was higher than the baseline (.23%, P < .001). Even after adjusting for subjects' age and gender, smoking history, and endoscopists' biopsy sampling rate, prolonged EGD observation time of more than 3 minutes increased the NDR of UGI neoplasms (odds ratio, 1.51; 95% confidence interval, 1.21-1.75). CONCLUSIONS: This study provides evidence that implementing a protocol of a prolonged observation time could increase NDR. Observation time should be an important quality indicator of the EGD examination.

Survival after in-hospital cardiopulmonary resuscitation from 2003 to 2013: An observational study before legislation on the life-sustaining treatment decision-making act of Korean patients.

We analyzed cardiopulmonary resuscitation (CPR) rates, deaths preceded by CPR, and survival trends after in-hospital CPR, using a sample of nationwide Korean claims data for the period 2003 to 2013.The Korean National Health Insurance Service-National Sample Cohort is a stratified random sample of 1,025,340 subjects selected from among approximately 46 million Koreans. We evaluated the annual incidence of CPR per 1000 admissions in various age groups, hospital deaths preceded by CPR, and survival rate following in-hospital CPR. Analyses of the relationships between survival and patient and hospital characteristics were performed using logistic regression analysis.A total of 5918 in-hospital CPR cases from 2003 to 2013 were identified among eligible patients. The cumulative incidence of in-hospital CPR was 3.71 events per 1000 admissions (95% confidence interval 3.62-3.80). The CPR rate per 1000 admissions was highest among the oldest age group, and the rate decreased throughout the study period in all groups except the youngest age group. Hospital deaths were preceded by in-hospital CPR in 18.1% of cases, and the rate decreased in the oldest age group. The survival-to-discharge rate in all study subjects was 11.7% during study period, while the 6-month and 1-year survival rates were 8.0% and 7.2%, respectively. Survival tended to increase throughout the study period; however, this was not the case in the oldest age group. Age and malignancy were associated with lower survival rates, whereas myocardial infarction and diabetes mellitus were associated with higher survival rates.Our result shows that hospital deaths were preceded by in-hospital CPR in 18.1% of case, and the survival-to-discharge rate in all study subjects was 11.7% during the study period. Survival tended to increase throughout the study period except for the oldest age group. Our results provide reliable data that can be used to inform judicious decisions on the implementation of CPR, with the ultimate goal of optimizing survival rates and resource utilization.

Reasons for Surgery Cancellation in a General Hospital: A 10-year Study.

Background: This study researched related causes that make scheduled surgeries canceled not to be conducted and based on the research it is to derive issues in order to reduce surgery cancellation. Methods: We analyzed the association of surgery cancellation with patient characteristics, surgical characteristics and surgery schedule related characteristics, using electronic medical record (EMR) data on surgeries conducted at a university hospital in Korea over 10 years. Additionally, we examined the reasons for surgery cancellation based on patient and hospital characteristics. We used chi-square tests to analyze the distribution of various characteristics according to reasons for surgery cancellation. Multivariate logistic regression analyses were conducted to evaluate the factors associated with surgery cancellation. Results: Among 60,333 cases, surgery cancellation rate was 8.0%. The results of the logistic regression indicated a high probability of surgery cancellation when the patient was too old (odds ratio [OR]: 1.35, 95% confidence interval [CI]: 1.14⁻1.59), when it was a neurosurgery case (OR: 1.39, 95% CI: 1.21⁻1.59), when local anesthesia was used (OR: 1.15, 95% CI: 1.07⁻1.24) or when it was a planned surgery (OR: 2.45, 95% CI: 2.21⁻2.73). The surgery cancellation rate was lower when the patient was female (OR: 0.87, 95% CI: 0.82⁻0.93) or when the surgery was related to Obstetrics & Gynecology (OR: 0.53, 95% CI: 0.46⁻0.60) or Ophthalmology (OR: 0.66, 95% CI: 0.56⁻0.79). Among the canceled 4834 cases, the surgery cancellation rate for the reasons of patients was 93.2% and the surgery cancellation rate for the reasons of a hospital was 6.8%. Conclusions: This study found that there are related various causes to cancel operations, including patient characteristics, surgery related characteristics and surgery schedule related characteristics and it means that it would be possible for some reasons to be prevented. Every medical institution should consider the operation cancellation as an important issue and systematic monitoring should be needed.

Dynamics of Quaternary Structure Transitions in R-State Carbonmonoxyhemoglobin Unveiled in Time-Resolved X-ray Scattering Patterns Following a Temperature Jump.

It is well-known that tetrameric hemoglobin binds ligands cooperatively by undergoing a ligand-induced T → R quaternary structure transition, a structure-function relationship that has long served as a model system for understanding allostery in proteins. However, kinetic studies of the reverse, R → T quaternary structure transition following photolysis of carbonmonoxyhemoglobin (HbCO) reveal complex behavior that may be better explained by the presence of two different R quaternary structures coexisting in thermal equilibrium. Indeed, we report here time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) patterns of HbCO following a temperature jump that not only provide unambiguous evidence for more than one R state, but also unveil the time scale for interconversion between them. Since the time scale for the photolysis-induced R → T transition is likely different for different R-states, this structural heterogeneity must be accounted for to properly explain the kinetic heterogeneity observed in time-resolved spectroscopic studies following photolysis of HbCO.

Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.

The capacity to respond to environmental changes is crucial to an organism's survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 Å) is significantly larger than that for the ground state (Rg = 14.7 Å) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the ß-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.

Association of serum calcium concentrations with fibrinogen and homocysteine in nondiabetic Korean subjects.

Considerable evidence shows that increased serum calcium levels are associated with metabolic disorders, cardiovascular disease, and increased mortality. This study investigated whether serum calcium, within a normal range, is significantly associated with serum fibrinogen and homocysteine, markers of increased cardiovascular disease risk in nondiabetic Korean subjects.A cross-sectional analysis was performed on 1096 subjects (mean age, 55.1 ±â€Š11.1 years; 36.1% women) undergoing a general health checkup. Serum biochemistry was analyzed including serum albumin-corrected calcium (Cac), insulin resistance (IR, using homeostasis model assessment [HOMA]), fibrinogen, and homocysteine.Compared with patients within the lowest Cac quartile, those with higher Cac levels had increased fibrinogen and homocysteine levels as well as an increased proportion of smoking, dyslipidemia, and HOMA-IR. Correlation analyses revealed linear relationships for Cac with fibrinogen and homocysteine in both genders. After adjustment for confounding factors, serum Cac was significantly associated with high fibrinogen (odds ratio [OR] for the highest vs the lowest quartile = 1.76, 95% confidence interval [CI] = 1.09-2.83, P = 0.02) and homocysteine (OR = 1.83, 95% CI = 1.07-3.11, P = 0.027). Multivariate regression models showed that Cac was linearly associated with fibrinogen (standardized ß = 0.14, P < 0.001) and homocysteine (standardized ß = 0.07, P = 0.009).High normal calcium concentrations were independently associated with increased levels of fibrinogen and homocysteine. Further investigation is needed to validate whether slightly increased calcium levels within the normal range indicate a higher risk of cardiovascular disease.

Serum bilirubin levels are inversely associated with PAI-1 and fibrinogen in Korean subjects.

OBJECTIVES: Oxidative stress may contribute to atherosclerosis and increased activation of the coagulation pathway. Bilirubin may reduce activation of the hemostatic system to inhibit oxidative stress, which would explain its cardioprotective properties shown in many epidemiological studies. This study investigated the association of serum bilirubin with fibrinogen and plasminogen activator inhibitor-1 (PAI-1), respectively. METHODS: A cross-sectional analysis was performed on 968 subjects (mean age, 56.0 ± 11.2 years; 61.1% men) undergoing a general health checkup. Serum biochemistry was analyzed including bilirubin subtypes, insulin resistance (using homeostasis model of assessment [HOMA]), C-reactive protein (CRP), fibrinogen, and PAI-1. RESULTS: Compared with subjects with a total bilirubin (TB) concentration of <10.0 µmol/L, those with a TB concentration of >17.1 µmol/L had a smaller waist circumference, a lower triglyceride level, a lower prevalence of metabolic syndrome, and decreased HOMA-IR and CRP levels. Correlation analysis revealed linear relationships of fibrinogen with TB and direct bilirubin (DB), whereas PAI-1 was correlated with DB. After adjustment for confounding factors, bilirubin levels were inversely associated with fibrinogen and PAI-1 levels, respectively. Multivariate regression models showed a negative linear relationship between all types of bilirubin and fibrinogen, whereas there was a significant linear relationship between PAI-1 and DB. CONCLUSIONS: High bilirubin concentrations were independently associated with low levels of fibrinogen and PAI-1, respectively. The association between TB and PAI-1 was confined to the highest TB concentration category whereas DB showed a linear association with PAI-1. Bilirubin may protect against the development of atherothrombosis by reducing the hemostatic response.

Probing anisotropic structure changes in proteins with picosecond time-resolved small-angle X-ray scattering.

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals and suggest that the early time structural dynamics in the crystalline state carry over to proteins in solution.

Physico-chemical characterization-based safety evaluation of nanocalcium.

Nano- and microcalcium provided from the KFDA were compared in terms of physico-chemical properties. Calcium samples were tested using EF-TEM and X-ray diffractometry to check for size/morphology and crystal formation, respectively. Two samples of nano- and microcalcium were selected for further evaluation by FE-SEM, DLS (nano-size, 200-500nm; agglomerate, >5 µm; micro-size, 1.5-30 µm), and electron spin resonance. Both samples were heterogeneous in size, existed as single crystal and aggregated form, and did not generate reactive oxygen species. The specific surface area of nano- and microcalcium measured by N2 Brunauere Emmette Teller method was 12.90±0.27 m(2)/g and 1.12±0.19 m(2)/g, respectively. Inductively coupled plasma optical emission spectrometry analysis revealed the release of 2-3 times more calcium ion from nano- compared to microcalcium at pH 5 and 7. Genotoxicity and acute single-dose and repeated-dose 14-day oral toxicity testing in SD rats performed to evaluate the safety of nanocalcium did not reveal toxicity. However, long-term monitoring will be required for an unequivocal conclusion. A nanocalcium dose of 1 g/kg is recommended as the maximum dose for repeated dose 13-week oral toxicity. Further studies could provide details of toxicity of nanocalcium on the repeated dose 13-week oral toxicity test.

Real-time tracking of CO migration and binding in the α and ß subunits of human hemoglobin via 150-ps time-resolved Laue crystallography.

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and ß subunits from 100 ps to 10 µs. The proximity of the B site in the ß subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (~300 µs-1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the ß subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the ß subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.

Genotoxicity, acute oral and dermal toxicity, eye and dermal irritation and corrosion and skin sensitisation evaluation of silver nanoparticles.

To clarify the health risks related to silver nanoparticles (Ag-NPs), we evaluated the genotoxicity, acute oral and dermal toxicity, eye irritation, dermal irritation and corrosion and skin sensitisation of commercially manufactured Ag-NPs according to the OECD test guidelines and GLP. The Ag-NPs were not found to induce genotoxicity in a bacterial reverse mutation test and chromosomal aberration test, although some cytotoxicity was observed. In acute oral and dermal toxicity tests using rats, none of the rats showed any abnormal signs or mortality at a dose level of ∼ 2000 mg/kg. Similarly, acute eye and dermal irritation and corrosion tests using rabbits revealed no significant clinical signs or mortality and no acute irritation or corrosion reaction for the eyes and skin. In a skin sensitisation test using guinea pigs, one animal (1/20) showed discrete or patchy erythema, thus Ag-NPs can be classified as a weak skin sensitiser.

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography.

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.

Bilateral accessory inferior turbinates and secondary middle turbinates.

Bifid inferior nasal turbinates-two inferior turbinates with a single root observed in the nasal cavity-are an extremely rare anatomical variation, especially when they are observed bilaterally and exist with other variations. We report a case of bilateral bifid inferior nasal turbinates with bilateral secondary middle turbinates. We discuss the findings of this rare condition and suggest an appropriate term-accessory inferior turbinate-to replace bilateral inferior turbinate by reason of its embryology.

Ileal stenosis occurred 3 months after blunt abdominal trauma.

We present a case of ileal stenosis with delayed presentation 3 months after car accident. Ileal stenosis after blunt abdominal trauma is a rare clinical entity. We present CT and small bowel series 3 months after trauma. Image showed segmental thickening of intestinal wall and proximal bowel dilation. At surgery, a stenotic bowel loop was adjacent to a fibrotic mesentery. Histological examination showed ulcers, inflammatory cells and fibroblasts infiltrated to the muscularis mucosae, submucosa, and mesentery. The most likely cause, supported by most authors, implicates an injury to the mesentery. Post-traumatic ischemic bowel stenosis may result from even small tears and contusions of mesentery. Posttraumatic intestinal stenosis should be included in the differential diagnosis in a patient with a history of blunt abdominal trauma and signs of intestinal obstruction.

Anxiety, depression and quality of life in patients with irritable bowel syndrome.

BACKGROUND/AIMS: There have been few Asian studies regarding anxiety and depression associated with irritable bowel syndrome (IBS). The aim of this study was to evaluate the frequency and importance of anxiety and depression in Korean patients with IBS. METHODS: A total of 124 IBS patients and 91 healthy subjects were enrolled consecutively. All participants were asked to complete self-administered questionnaires: one addressing symptom severity, the Short Form 36, and the Hospital Anxiety and Depression Scale (HADS). The patients were also asked to complete the IBS-specifi c quality of life (IBS-QOL) questionnaire. RESULTS: Anxiety and depression were observed in 38.6% and 38.6% of IBS patients, respectively, and in 24.2% and 16.5% of healthy subjects, respectively (p<0.05 for both). The mean HADS scores for anxiety and depression in IBS patients were 6.8±4.5 and 7.1±4.4, respectively. Both anxiety and depression were associated with self-reported symptom severity (p<0.012 and p<0.001, respectively). As determined by multivariate analysis, symptom severity was the most important factor in the prediction of anxiety and depression. Self-reported symptom severity and depression were clearly and independently associated with the overall IBS-QOL score. CONCLUSIONS: Anxiety and depression were frequently observed in Korean IBS patients and were related to the severity of their symptoms and the impairment of the patient's QOL. Our data suggest that assessing anxiety and depression is important when evaluating IBS patients.

Aspect ratio has no effect on genotoxicity of multi-wall carbon nanotubes.

Carbon nanotubes (CNTs) have specific physico-chemical and electrical properties that are useful for telecommunications, medicine, materials, manufacturing processes and the environmental and energy sectors. Yet, despite their many advantages, it is also important to determine whether CNTs may represent a hazard to the environment and human health. Like asbestos, the aspect ratio (length:diameter) and metal components of CNTs are known to have an effect on the toxicity of carbon nanotubes. Thus, to evaluate the toxic potential of CNTs in relation to their aspect ratio and metal contamination, in vivo and in vitro genotoxicity tests were conducted using high-aspect-ratio (diameter: 10-15 nm, length: ~10 µm) and low-aspect-ratio multi-wall carbon nanotubes (MWCNTs, diameter: 10-15 nm, length: ~150 nm) according to OECD test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test), and 474 (in vivo micronuclei test) with a good laboratory practice system. To determine the treatment concentration for all the tests, a solubility and dispersive test was performed, and a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) solution found to be more suitable than distilled water. Neither the high- nor the low-aspect-ratio MWCNTs induced any genotoxicity in a bacterial reverse mutation test (~1,000 µg/plate), in vitro chromosome aberration test (without S9: ~6.25 µg/ml, with S9: ~50 µg/ml), or in vivo micronuclei test (~50 mg/kg). However, the high-aspect-ratio MWCNTs were found to be more toxic than the low-aspect-ratio MWCNTs. Thus, while high-aspect-ratio MWCNTs do not induce direct genotoxicity or metabolic activation-mediated genotoxicity, genotoxicity could still be induced indirectly through oxidative stress or inflammation.