Assessing the Change in Operative Treatment Rates for Acute Midshaft Clavicle Fractures: Incorporation of Evidence-Based Surgery Results in Orthopaedic Practice.

In 2007, a randomized controlled trial (RCT) by the Canadian Orthopaedic Trauma Society (COTS) demonstrated better functional outcomes and a lower proportion of patients who developed malunion or nonunion following operative, compared with nonoperative, treatment of midshaft clavicle fractures. The primary aim of the present study was to compare the proportion of midshaft clavicle fractures treated operatively prior to and following the publication of the COTS RCT. An additional exploratory aim was to assess whether the proportion of midshaft clavicle fractures that were treated with surgery for malunion or nonunion decreased. Methods: This retrospective cohort analysis used population-level administrative health data on the residents of British Columbia, Canada. Cases were identified by International Classification of Diseases, Ninth Revision (ICD-9) diagnostic codes and procedure fee codes. Adult patients (≥18 years) with closed middle-third clavicle fractures between 1997 and 2018 were included. Multivariable logistic regression modeling compared the proportion of clavicle fractures treated operatively before and after January 1, 2007, controlling for patient factors. The Pearson chi-square test compared the proportion of fractures treated operatively for malunion or nonunion in the cohorts. Results: A total of 52,916 patients were included (mean age, 47.5 years; 65.6% male). More clavicle fractures were treated operatively from 2007 onward: 6.9% compared with 2.2% prior to 2007 (odds ratio [OR] = 3.35, 95% confidence interval [CI] = 3.03 to 3.70, p < 0.001). Male sex, moderate-to-high income, and younger age were associated with a greater proportion of operative fixation. The rate of surgery for clavicle malunion or nonunion also increased over this time period (to 4.1% from 3.4%, OR = 1.26, 95% CI = 1.15 to 1.38, p < 0.001). Conclusions: We found a significant change in surgeon practice regarding operative management of clavicle fractures following the publication of a Level-I RCT. With limited high-quality trials comparing operative and nonoperative management, it is important that clinicians, health-care institutions, and health-authority administrations determine what steps can be taken to increase responsiveness to new clinical studies and evidence-based guidelines. Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Use of intraoperative impression smear cytology to guide successful treatment of a large renal cyst in a dog: a case report.

A 13-yr-old Shih tzu was referred for surgical management of right-sided cranial abdominal mass, which corresponded to large, cavitated renal mass on ultrasonography, and was suspected to represent neoplasia. Intraoperative impression smear cytology (ISC) of the renal mass wall was consistent with benign renal cyst (RC), without evidence of neoplasia or infection. Deroofing and omentalisation were performed and histopathology was consistent with benign RC. Chronic kidney disease was diagnosed 4 mon postoperatively, however, the dog was asymptomatic, without cyst reoccurrence. Intraoperative ISC is an expedient and inexpensive diagnostic technique that can guide most appropriate treatment in dogs with large RCs.

A head-to-head Caco-2 assay comparison of the mechanisms of action of the intestinal permeation enhancers: SNAC and sodium caprate (C10).

Salcaprozate sodium (SNAC) and sodium caprate (C10) are the two leading intestinal permeation enhancers (PEs) in oral peptide formulations in clinical trials. There is debate over their mechanism of action on intestinal epithelia. The aims were: (i) to compare their effects on the barrier function by measuring transepithelial electrical resistance (TEER), permeability of FITC-4000 (FD4) across Caco-2 monolayers, and on immunohistochemistry of tight junction (TJ)-associated proteins; and (ii) to compare cellular parameters using conventional end-point cytotoxicity assays and quantitative high content analysis (HCA) of multiple sub-lethal parameters in Caco-2 cells. C10 (8.5 mM) reversibly reduced TEER and increased FD4 permeability across monolayers, whereas SNAC had no effects on either parameter except at cytotoxic concentrations. C10 exposure induced reorganization of three TJ proteins, whereas SNAC only affected claudin-5 localization. High concentrations of C10 and SNAC were required to cause end-point toxicology changes in vitro. SNAC was less potent than C10 at inducing lysosomal and nuclear changes and plasma membrane perturbation. In parallel, HCA revealed that both agents displayed detergent-like features that reflect initial membrane fluidization followed by changes in intracellular parameters. In conclusion, FD4 permeability increases in monolayers in response to C10 were in the range of concentrations that altered end-point cytotoxicity and HCA parameters. For SNAC, while HCA parameters were also altered in a similar overall pattern as C10, they did not lead to increased paracellular flux. These assays show that both agents are primarily surfactants, but C10 has additional TJ-opening effects. While these in vitro assays illucidate their epithelial mechanism of action, clinical experience suggests that they over-estimate their toxicology in the dynamic intestinal environment.

Measuring Outcomes Over Time in Distal Radius Fractures: A Comparison of Generic, Upper Extremity-Specific and Wrist-Specific Outcome Measures.

Purpose: This study compared the responsiveness of a generic (Short Form-36 [SF-36]), an upper extremity-specific (Disabilities of the Arm, Shoulder, and Hand [DASH]) and a wrist-specific (Patient-Rated Wrist Evaluation [PRWE]) outcome score when evaluating distal radius fractures over time. Methods: We observed 235 patients who met the inclusion criteria of an isolated distal radius fracture treated surgically or nonsurgically and greater than age 50 years for 12 months in this prospective study. Standardized assessments were performed at baseline and at 6 and 12 months. Exclusion criteria included subjects with concomitant injuries in the ipsilateral limb and follow-up of less than 1 year. Responsiveness was evaluated through the standardized response mean and the proportion who met a minimal clinically important difference. Floor and ceiling effects were also calculated. Results: The standardized response mean was significantly greatest for the DASH between baseline and 6 months (P < .001), and the PRWE between both baseline and 6 months (P < .01) and 6 and 12 months (P < .01) compared with the SF-36. The proportion of patients who met a minimal clinically important difference between baseline and 6 months was greater in the PRWE, but it did not meet statistical significance (P = .12). The PRWE demonstrated a high ceiling effect at baseline (76.6%) but less so at 12 months (16.9%). The DASH demonstrated similar ceiling effects at baseline (62.9%) and 12 months (18.6%). The SF-36 had no ceiling effect. Conclusions: In the first 6 months, both the DASH and PRWE have greater responsiveness in assessing change over the SF-36 in distal radius fractures. From 6 to 12 months, the wrist-specific PRWE has greater responsiveness over both the DASH and SF-36. This supports the use of the anatomy- and injury-specific outcome measures over the generic outcome measure in detecting change over a patient's early recovery. However, as the time from injury increases, the absence of a ceiling effect from the generic outcome measure may become more useful. Clinical relevance: This study demonstrates the responsiveness of the DASH, PRWE, and SF36 in assessing distal radius fractures treated in patients greater than age 50 in the first year. In establishing the most responsive measure, respondent burden can be decreased in future research.

The Feasibility of a Randomized Controlled Trial for Open Tibial Fractures at a Regional Hospital in Uganda.

BACKGROUND: The inclusion of low and middle-income country (LMIC) hospitals in multicenter orthopaedic trials expands the pool of eligible patients and improves the external validity of the evidence. Furthermore, promoting studies in LMIC hospitals defines the optimal treatments for low-resource settings, the conditions under which the majority of musculoskeletal injuries are treated. The objective of this study was to determine the feasibility of a randomized controlled trial comparing external fixation with intramedullary (IM) nailing in patients with an isolated open tibial fracture who presented to a regional hospital in Uganda. METHODS: From July 2016 to July 2017, skeletally mature patients who presented to a Ugandan regional hospital with an isolated Gustilo-Anderson type-II or IIIA open fracture of the tibial shaft were eligible for inclusion. The primary feasibility outcomes were the enrollment rate, the recruitment rate, and the 3 and 12-month follow-up rates. The secondary outcomes included a comparison of 3 and 12-month follow-up rates between the treatment arms and a qualitative assessment of barriers to enrollment, timely treatment, and missed follow-up. RESULTS: During the 12-month enrollment period, 37.5% (30 of 80) of eligible patients were successfully enrolled and operatively treated on the basis of their random allocation, with an enrollment rate of 2.5 patients per month. Of the 30 enrolled patients, 53% completed their 3-month follow-up appointment, and 40% completed their 1-year follow-up appointment. Rates of 1-year follow-up were significantly higher for patients receiving IM nails than for those receiving external fixation (absolute difference, 52%; 95% confidence interval [CI], 21 to 83, p < 0.01). The main reasons that patients declined to participate in the trial were preferences for treatment by traditional bonesetters and prehospital delays that were related to a disorganized referral system. Barriers to follow-up included prohibitive transportation costs and community pressure to turn to traditional forms of treatment. CONCLUSIONS: A regional hospital in Uganda can successfully enroll, randomize, and operatively treat multiple patients with an open tibial fracture each month. Patient follow-up presents substantial concerns over trial feasibility in this setting. Cultural pressure to utilize traditional treatments remains a particularly common barrier to study-participant enrollment and retention.

High rate of reoperation and conversion to total hip arthroplasty after internal fixation of young femoral neck fractures: a population-based study of 796 patients.

Background and purpose - Most often, the goal of non-geriatric femoral neck fracture surgery is to preserve the native hip joint. However, reoperations for painful implants, osteonecrosis, and nonunion are common. We determined the reoperation rate and time-to-reoperation following internal fixation of these fractures in a large population cohort. Patients and methods - This retrospective cohort study included patients between the ages of 18 and 50 years old who underwent internal fixation for a femoral neck fracture during 1997-2013. Patients were followed until December 2013. Primary outcomes were reoperation rate and time-to-reoperation. Time-to-event analysis was performed to estimate the rate of any reoperation and for THA specifically, while testing the dependency of time-to-reoperation on secondary variables. Results - 796 young femoral neck fracture patients were treated with internal fixation during the study period (median age 43 years, 39% women). Median follow-up was 8 years (IQR 4-13). One-third underwent at least 1 reoperation at a median 16 months after the index surgery (IQR 8-31). Half of reoperations were for implant removal, followed by conversion to total hip arthroplasty. 14% of the cohort were converted to THA. The median time to conversion was 2 years (IQR 1-4). Neither female sex nor older age had a statistically significant effect on time-to-reoperation or time-to-THA conversion. Interpretation - Following internal fixation of young femoral neck fracture, 1 in 3 patients required a reoperation, and 1 in 7 were converted to THA. These data should be considered by patients and surgeons during treatment decision-making.

Predicting completion of follow-up in prospective orthopaedic trauma research.

OBJECTIVE: Orthopaedic trauma studies that collect long-term outcomes are expensive and maintaining high rates of follow-up can be challenging. Knowing what factors influence completion of follow-up could allow interventions to improve this. We aimed to assess which factors influence completion of follow-up in the 12 months following surgery in prospective orthopaedic trauma research. DESIGN: Prospective Cohort Study. SETTING: Level 1 Trauma Center, Vancouver, Canada. PARTICIPANTS: Eight hundred seventy patients recruited to 4 prospective studies investigating the outcomes of operatively treated lower extremity fractures. MAIN OUTCOME MEASUREMENTS: Completion of follow-up defined as completion of all outcome measures at all time points up to 12 months following injury. RESULTS: Univariate analysis and subsequent analysis by building a reductive multivariate regression model allowed for estimation of the influence of factors in completion of follow-up.Eight hundred seventy patients with complete data had previously been recruited and were included in the analysis. Seven hundred seven patients (81.2%) completed follow-up to 12 months. Factors associated with completion of follow up included higher physical component score of SF-36 at baseline, not being on social assistance at the time of injury, being married and having a higher level of educational attainment. CONCLUSIONS: Our study has demonstrated several important factors identifiable at baseline which are associated with a failure to complete follow-up. Although these factors are not modifiable themselves, we advocate that researchers designing studies should plan for additional follow-up resources and interventions for at risk patients. LEVEL OF EVIDENCE: Level IV.

Comparison of Generic, Musculoskeletal-Specific, and Foot and Ankle-Specific Outcome Measures Over Time in Tibial Plafond Fractures.

BACKGROUND: This study performed a psychometric analysis assessing and comparing the responsiveness of the relevant components of a generic (Short Form-36 [SF36]), a musculoskeletal-specific (Short Musculoskeletal Functional Assessment [SMFA]), and a foot and ankle-specific (Foot and Ankle Outcome Score [FAOS]) outcome score when evaluating surgically treated tibial plafond fractures over time. METHODS: Fifty-one patients were followed for 12 months after their tibial plafond fracture. Responsiveness, or the ability to detect clinical change in a disease, was evaluated through the standardized response mean (SRM), the proportion meeting a minimal clinically important difference (MCID), and floor and ceiling effects. RESULTS: The SRM of the SF36-Physical Component Summary (PCS) was significantly greater than the SMFA-dysfunction index (DI) (P < .01) and FAOS-Activities of Daily Living (ADL) (P = .01) between baseline and 6 months, whereas the SRMs of only SF36-PCS and FAOS-ADL differed (P = .01) between 6 and 12 months. The proportion of patients achieving an MCID for SF36-PCS was higher than FAOS-ADL (P = .03) between baseline and 6 months and higher than SMFA-DI (P = .04) between 6 and 12 months. The FAOS-ADL showed substantial ceiling effects at baseline (88.2%) but much less at 6 months (5.9%) and 12 months (9.8%). Smaller ceiling effects were observed for the SMFA-DI (11.8%) at baseline, whereas none were observed for the SF36-PCS. CONCLUSIONS: This study found that the SF36-PCS had greater responsiveness in assessing tibial plafond fractures compared to the SMFA-DI and FAOS-ADL, particularly in the first 6 months after surgery. In addition, limitations were revealed in the SMFA-DI and FAOS-ADL. This study illustrates the necessary diligence required for selection of outcome measures, as musculoskeletal and anatomy specific scores are not necessarily superior. LEVEL OF EVIDENCE: Level II, prospective cohort study.

The Socioeconomic Implications of Isolated Tibial and Femoral Fractures from Road Traffic Injuries in Uganda.

BACKGROUND: The purpose of this study was to determine the socioeconomic implications of isolated tibial and femoral fractures caused by road traffic injuries in Uganda. METHODS: This prospective longitudinal study included adult patients who were admitted to Uganda's national referral hospital with an isolated tibial or femoral fracture. The primary outcome was the time to recovery following injury. We assessed recovery using 4 domains: income, employment status, health-related quality of life (HRQoL) recovery, and school attendance of the patients' dependents. RESULTS: The majority of the study participants (83%) were employed, and they were the main income earner for their household (74.0%) at the time of injury, earning a mean annual income of 2,375 U.S. dollars (USD). All of the patients had been admitted with the intention of surgical treatment; however, because of resource constraints, only 56% received operative treatment. By 2 years postinjury, only 63% of the participants had returned to work, and 34% had returned to their previous income level. Overall, the mean monthly income was 62% less than preinjury earnings, and participants had accumulated 1,069 USD in debt since the injury; 41% of the participants had regained HRQoL scores near their baseline, and 62% of school-aged dependents, enrolled at the time of injury, were in school at 2 years postinjury. CONCLUSIONS: At 2 years postinjury, only 12% of our cohort of Ugandan patients who had sustained an isolated tibial or femoral fracture from a road traffic injury had recovered both economically and physically. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Methotrexate induced mitochondrial injury and cytochrome c release in rat liver hepatocytes.

Methotrexate (MTX) is a folic acid antagonist that is widely used to treat a variety of diseases. One of the most serious side effects of MTX therapy is hepatotoxicity. The potential molecular cytotoxic mechanisms of MTX toward isolated rat hepatocytes were investigated using Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques. A concentration and time dependent increase in cytotoxicity and reactive oxygen species (ROS) formation and a decrease in mitochondrial membrane potential (MMP) were observed with MTX. Furthermore, a significant increase in MTX (300 µM)-induced cytotoxicity and ROS formation were observed when glutathione (GSH)-depleted hepatocytes were used whereas addition of N-acetylcysteine (a GSH precursor) decreased cytotoxicity. Catalase inactivation also increased MTX-induced cytotoxicity, while the direct addition of catalase to the hepatocytes decreased cytotoxicity. MTX treatment in isolated rat mitochondria caused swelling and significantly decreased adenosine triphosphate (ATP) and GSH content, and cytochrome c release. Potent antioxidants such as mesna, resveratrol and Trolox decreased MTX-induced cytotoxicity and ROS formation and increased MMP. This study suggests that MTX-induced cytotoxicity caused by ROS formation and GSH oxidation leads to oxidative stress and mitochondrial injury in rat hepatocytes.

Marked paraneoplastic basophilia accompanying eosinophilia in a cat with alimentary T-cell lymphoma.

CASE SUMMARY: A 5-year-old male neutered domestic shorthair cat was referred with a history of persistent pyrexia, pica, soft faeces, inappetence, intermittent vomiting, mild-to-moderate granulocytosis and mild hypercalcaemia. No significant improvement was noted after antibiotic and corticosteroid treatment, except that the hypercalcaemia resolved. Physical examination, including thoracic auscultation, and abdominal and peripheral lymph node palpation, were unremarkable. On admission, haematology revealed moderate leukocytosis (36.8 × 109/l) with moderate-to-marked eosinophilia (21.3 × 109/l) and marked basophilia (4.04 × 109/l), the latter identified microscopically. Lymphocytes were markedly decreased (0.37 × 109/l). Blood smear examination revealed 58% eosinophils, 28% neutrophils, 11% basophils, 2% monocytes, 1% lymphocytes and marked, diffuse platelet clumping. Biochemistry abnormalities indicated mild pancreatitis, dehydration and anorexia with mildly increased pancreatic lipase, mild hypernatraemia (157 mmol/l), a moderate decrease in urea (3.1 mmol/l) and a slight decrease in phosphate (1.32 mmol/l). Ultrasound and radiographic imaging revealed enlargement of the mesenteric lymph nodes. Fine-needle aspiration, a Tru-cut biopsy and immunohistochemistry were performed. Cytological examination revealed ~65-75% lymphocytes (~80% were larger than a neutrophil), ~25-35% eosinophils and occasional basophils. Lymphocytes had single, small (<1/3 red blood cells), prominent nucleoli and increased pale, mildly vacuolated cytoplasm. On histopathology, cells were monomorphic, large, with prominent nucleoli, and mild, multifocal, staining for T-cell marker CD3. Smaller cells were strongly CD3-positive. Cells were negative for B-cell marker CD45R. RELEVANCE AND NOVEL INFORMATION: This is the most severe case of paraneoplastic basophilia reported with feline alimentary T-cell lymphoma with accompanying eosinophilia and lymph node infiltration. Feline basophil prevalence is reported for the first time.

When Surgical Resources are Severely Constrained, Who Receives Care? Determinants of Access to Orthopaedic Trauma Surgery in Uganda.

BACKGROUND: In low- and middle-income countries, the volume of traumatic injuries requiring orthopaedic intervention routinely exceeds the capacity of available surgical resources. The objective of this study was to identify predictors of surgical care for lower extremity fracture patients at a high-demand, resource-limited public hospital in Uganda. METHODS: Skeletally mature patients admitted with the intention of definitive surgical treatment of an isolated tibia or femur fractures to the national referral hospital in Uganda were recruited to participate in this study. Demographic, socioeconomic, and clinical data were collected through participant interviews at the time of injury and 6 months post-injury. Social capital (use of social networks to gain access to surgery), financial leveraging, and ethnicity were also included as variables in this analysis. A probit estimation model was used to identify independent and interactive predictors of surgical treatment. RESULTS: Of the 64 patients included in the final analysis, the majority of participants were male (83%), with a mean age of 40.6, and were injured in a motor vehicle accident (77%). Due to resource constraints, only 58% of participants received surgical care. The use of social capital and femur fractures were identified as significant predictors of receiving surgical treatment, with social capital emerging as the strongest predictor of access to surgery (p < 0.05). CONCLUSION: Limited infrastructure, trained personnel, and surgical supplies rations access to surgical care. In this environment, participants with advantageous social connections were able to self-advocate for surgery where demand for these services greatly exceeded available resources.

Risk factors for colorectal cancer in man induce aberrant crypt foci in rats: Preliminary findings.

Epidemiological studies have demonstrated clear associations between specific dietary and environmental risk factors and incidence of colorectal cancer, but the mechanisms responsible for these associations are not known. An animal model could facilitate such an understanding. Both genotoxic and nongenotoxic carcinogens induce aberrant crypt foci (ACF) in the colons of F344 rats. F344 rats were provided with diets that contained putative risk factors for CRC: low calcium and low vitamin D, high iron, high fructose, and decreased light (UV) exposure or a control diet for 14 wk. The rats were then assessed with biochemical measures and by topological examination for evidence of colon abnormalities. Circulating ionized calcium was decreased from 2.85 to 1.69 mmol/L, and ACF were increased from 0.7 to 13.6 lesions/colon (both P < 0.001). Rats exposed to the multiple environmental conditions associated with colon cancer, developed ACF similar to the heterogeneous or ill-defined ACF in the human colon. Heterogeneous ACF are the most frequently seen in humans and are also seen in rats shortly after exposure to the non-genotoxic colon carcinogen, dextransulfate sodium. The rodent model could be used to assess the pathways from diet and environment to colon cancer and to provide guidance for clinical studies.

The Association between Glyceraldehyde-Derived Advanced Glycation End-Products and Colorectal Cancer Risk.

BACKGROUND: A large proportion of colorectal cancers are thought to be associated with unhealthy dietary and lifestyle exposures, particularly energy excess, obesity, hyperinsulinemia, and hyperglycemia. It has been suggested that these processes stimulate the production of toxic reactive carbonyls from sugars such as glyceraldehyde. Glyceraldehyde contributes to the production of a group of compounds known as glyceraldehyde-derived advanced glycation end-products (glycer-AGEs), which may promote colorectal cancer through their proinflammatory and pro-oxidative properties. The objective of this study nested within a prospective cohort was to explore the association of circulating glycer-AGEs with risk of colorectal cancer. METHODS: A total of 1,055 colorectal cancer cases (colon n = 659; rectal n = 396) were matchced (1:1) to control subjects. Circulating glycer-AGEs were measured by a competitive ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (95% CI), adjusting for potential confounding factors, including smoking, alcohol, physical activity, body mass index, and diabetes status. RESULTS: Elevated glycer-AGEs levels were not associated with colorectal cancer risk (highest vs. lowest quartile, 1.10; 95% CI, 0.82-1.49). Subgroup analyses showed possible divergence by anatomical subsites (OR for colon cancer, 0.83; 95% CI, 0.57-1.22; OR for rectal cancer, 1.90; 95% CI, 1.14-3.19; Pheterogeneity = 0.14). CONCLUSIONS: In this prospective study, circulating glycer-AGEs were not associated with risk of colon cancer, but showed a positive association with the risk of rectal cancer. IMPACT: Further research is needed to clarify the role of toxic products of carbohydrate metabolism and energy excess in colorectal cancer development.

Protective effects of ferulic acid and related polyphenols against glyoxal- or methylglyoxal-induced cytotoxicity and oxidative stress in isolated rat hepatocytes.

Glyoxal (GO) and methylglyoxal (MGO) cause protein and nucleic acid carbonylation and oxidative stress by forming reactive oxygen and carbonyl species which have been associated with toxic effects that may contribute to cardiovascular disease, complications associated with diabetes mellitus, Alzheimer's and Parkinson's disease. GO and MGO can be formed through oxidation of commonly used reducing sugars e.g., fructose under chronic hyperglycemic conditions. GO and MGO form advanced glycation end products which lead to an increased potential for developing inflammatory diseases. In the current study, we have investigated the protective effects of ferulic acid and related polyphenols e.g., caffeic acid, p-coumaric acid, methyl ferulate, ethyl ferulate, and ferulaldehyde on GO- or MGO-induced cytotoxicity and oxidative stress (ROS formation, protein carbonylation and mitochondrial membrane potential maintenance) in freshly isolated rat hepatocytes. To investigate and compare the protective effects of ferulic acid and related polyphenols against GO- or MGO-induced toxicity, five hepatocyte models were used: (a) control hepatocytes, (b) GSH-depleted hepatocytes, (c) catalase-inhibited hepatocytes, (d) aldehyde dehydrogenase (ALDH2)-inhibited hepatocytes, and (e) hepatocyte inflammation system (a non-toxic H2O2-generating system). All of the polyphenols tested significantly decreased GO- or MGO-induced cytotoxicity, ROS formation and improved mitochondrial membrane potential in these models. The rank order of their effectiveness was caffeic acid∼ferulaldehyde>ferulic acid>ethyl ferulate>methyl ferulate>p-coumaric acid. Ferulic acid was found to decrease protein carbonylation in GSH-depleted hepatocytes. This study suggests that ferulic acid and related polyphenols can be used therapeutically to inhibit or decrease GO- or MGO-induced hepatotoxicity.

High content analysis provides mechanistic insights on the pathways of toxicity induced by amine-modified polystyrene nanoparticles.

The fast-paced development of nanotechnology needs the support of effective safety testing. We have developed a screening platform measuring simultaneously several cellular parameters for exposure to various concentrations of nanoparticles (NPs). Cell lines representative of different organ cell types, including lung, endothelium, liver, kidney, macrophages, glia, and neuronal cells were exposed to 50 nm amine-modified polystyrene (PS-NH2) NPs previously reported to induce apoptosis and to 50 nm sulphonated and carboxyl-modified polystyrene NPs that were reported to be silent. All cell lines apart from Raw 264.7 executed apoptosis in response to PS-NH2 NPs, showing specific sequences of EC50 thresholds; lysosomal acidification was the most sensitive parameter. Loss of mitochondrial membrane potential and plasma membrane integrity measured by High Content Analysis resulted comparably sensitive to the equivalent OECD-recommended assays, allowing increased output. Analysis of the acidic compartments revealed good cerrelation between size/fluorescence intensity and dose of PS-NH2 NPs applied; moreover steatosis and phospholipidosis were observed, consistent with the lysosomal alterations revealed by Lysotracker green; similar responses were observed when comparing astrocytoma cells with primary astrocytes. We have established a platform providing mechanistic insights on the response to exposure to nanoparticles. Such platform holds great potential for in vitro screening of nanomaterials in highthroughput format.

Evaluation of azathioprine-induced cytotoxicity in an in vitro rat hepatocyte system.

Azathioprine (AZA) is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using "Accelerated Cytotoxicity Mechanism Screening" techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 µM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS) formation was observed when glutathione- (GSH-) depleted hepatocytes were used. The addition of N-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2) formation and increased % mitochondrial membrane potential (MMP). Addition of N-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N'-diphenyl-p-phenylenediamine), Trolox (a water soluble vitamin E analogue), and mesna (2-mercaptoethanesulfonate), also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury.

Luciferase does not Alter Metabolism in Cancer Cells.

Luciferase transfected cell lines are used extensively for cancer models, revealing valuable biological information about disease mechanisms. However, these genetically encoded reporters, while useful for monitoring tumor response in cancer models, can impact cell metabolism. Indeed firefly luciferase and fatty acyl-CoA synthetases differ by a single amino acid, raising the possibility that luciferase activity might alter metabolism and introduce experimental artifacts. Therefore knowledge of the metabolic response to luciferase transfection is of significant importance, especially given the thousands of research studies using luciferase as an in vivo bioluminescence imaging (BLI) reporter. Untargeted metabolomics experiments were performed to examine three different types of lymphoblastic leukemia cell lines (Ramos, Raji and SUP T1) commonly used in cancer research, each were analyzed with and without vector transduction. The Raji model was also tested under perturbed starvation conditions to examine potential luciferase-mediated stress responses. The results showed that no significant metabolic differences were observed between parental and luciferase transduced cells for each cell line, and that luciferase overexpression does not alter cell metabolism under basal or perturbed conditions.

High-content analysis in toxicology: screening substances for human toxicity potential, elucidating subcellular mechanisms and in vivo use as translational safety biomarkers.

High-content analysis (HCA) of in vitro biochemical and morphological effects of classic (small molecule) drugs and chemicals is concordant with potential for human toxicity. For hepatotoxicity, concordance is greater for cytotoxic effects assessed by HCA than for conventional cytotoxicity tests and for regulatory animal toxicity studies. Additionally, HCA identifies chronic toxicity potential, and drugs producing idiosyncratic adverse reactions and/or toxic metabolites are also identified by HCA. Mechanistic information on the subcellular basis for the toxicity is frequently identified, including various mitochondrial effects, oxidative stress, calcium dyshomeostasis, phospholipidosis, apoptosis and antiproliferative effects, and a fingerprinting of the sequence and pattern of subcellular events. As these effects are frequently non-specific and affect many cell types, some toxicities may be detected and monitored by HCA of peripheral blood cells, such as for anticancer and anti-infective drugs. Critical methodological and interpretive features are identified that are critical to the effectiveness of the HCA cytotoxicity assessment, including the need for multiple days of exposure of cells to drug, use of a human hepatocyte cell line with metabolic competence, assessment of multiple pre-lethal effects in individual live cells, consideration of hormesis, the need for interpretation of relevance of cytotoxicity concentration compared to efficacy concentration and quality management. Limitations of the HCA include assessment of drugs that act on receptors, transporters or processes not found in hepatocytes. HCA may be used in a) screening lead candidates for potential human toxicity in drug discovery alongside of in vitro assessment of efficacy and pharmacokinetics, b) elucidating mechanisms of toxicity and c) monitoring in vivo toxicity of drugs with known toxicity of known mechanism.

Evaluation of the cytotoxicity of organic dust components on THP1 monocytes-derived macrophages using high content analysis.

Organic dust contains pathogen-associated molecular patterns (PAMPs) which can induce significant airway diseases following chronic exposure. Mononuclear phagocytes are key protecting cells of the respiratory tract. Several studies have investigated the effects of PAMPs and mainly endotoxins, on cytokine production. However the sublethal cytotoxicity of organic dust components on macrophages has not been tested yet. The novel technology of high content analysis (HCA) is already used to assess subclinical drug-induced toxicity. It combines the capabilities of flow cytometry, intracellular fluorescence probes, and image analysis and enables rapid multiple analyses in large numbers of samples. In this study, HCA was used to investigate the cytotoxicity of the three major PAMPs contained in organic dust, i.e., endotoxin (LPS), peptidoglycan (PGN) and ß-glucans (zymosan) on THP-1 monocyte-derived macrophages. LPS was used at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1, and 1 µg/mL; PGN and zymosan were used at concentrations of 1, 5, 10, 50, 100, and 500 µg/mL. Cells were exposed to PAMPs for 24 h. In addition, the oxidative burst and the phagocytic capabilities of the cells were tested. An overlap between PGN intrinsic fluorescence and red/far-red fluorescent dyes occurred, rendering the evaluation of some parameters impossible for PGN. LPS induced sublethal cytotoxicity at the lowest dose (from 50 ng/mL). However, the greatest cytotoxic changes occurred with zymosan. In addition, zymosan, but not LPS, induced phagosome maturation and oxidative burst. Given the fact that ß-glucans can be up to 100-fold more concentrated in organic dust than LPS, these results suggest that ß-glucans could play a major role in macrophage impairment following heavy dust exposure and will merit further investigation in the near future.