Correction: Rehnelt et al. Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation. Int. J. Mol. Sci. 2017, 18, 2634.

The authors wish to make the following corrections to this paper [...].

[Analysis of the prognostic factors influencing the time elapsing until the contralateral hip fracture]. / Az ellenoldali csípotáji törésig eltelt idot befolyásoló prognosztikai tényezok vizsgálata.

INTRODUCTION: Although several national studies reported on the risk factors for contralateral hip fracture, there are no data about the prognostic factors of the time until contralateral hip fractures. AIM: The aim of the study was to analyse the impact of different prognostic factors on the time until the development of contralateral fracture and to determine the incidence of contralateral hip fractures after femoral neck fractures. METHOD: Patients aged 60 years and over with contralateral hip fracture between 01 Jan 2000 and 31 Dec 2008 were identified among those who suffered their femoral neck fracture in Hungary in 2000. Risk factors as age, sex, comorbidities, type of fracture and surgery, place of living and hospitals providing treatment for primary fracture were analysed by one way ANOVA focusing on the time until the development of contralateral hip fracture. RESULTS: 312 patients met the inclusion criteria. The incidence of contralateral hip fracture after femoral neck fracture ranged between 1.5% and 2.1%, the cumulative incidence was 8.24%. The mean time until the development of contralateral hip fracture was 1159.8 days. The incidence of contralateral hip fracture showed no significant deviation. Significantly shorter time (p = 0.010) was detected until the contralateral hip fracture in older patients with femoral neck fracture. CONCLUSIONS: The yearly incidence of contralateral hip fracture showed no significant difference by patients with femoral neck fracture over 60 years. The shorter time until the contralateral hip fracture by the older age groups highlights the need of elaboration of prevention strategies. Orv Hetil. 2018; 159(38): 1543-1547.

Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation.

Side effects on cardiac ion channels causing lethal arrhythmias are one major reason for drug withdrawals from the market. Field potential (FP) recording from cardiomyocytes, is a well-suited tool to assess such cardiotoxic effects of drug candidates in preclinical drug development, but it is currently limited to the spontaneous beating of the cardiomyocytes and manual analysis. Herein, we present a novel optogenetic cardiotoxicity screening system suited for the parallel automated frequency-dependent analysis of drug effects on FP recorded from human-induced pluripotent stem cell-derived cardiomyocytes. For the expression of the light-sensitive cation channel Channelrhodopsin-2, we optimised protocols using virus transduction or transient mRNA transfection. Optical stimulation was performed with a new light-emitting diode lid for a 96-well FP recording system. This enabled reliable pacing at physiologically relevant heart rates and robust recording of FP. Thereby we detected rate-dependent effects of drugs on Na⁺, Ca2+ and K⁺ channel function indicated by FP prolongation, FP shortening and the slowing of the FP downstroke component, as well as generation of afterdepolarisations. Taken together, we present a scalable approach for preclinical frequency-dependent screening of drug effects on cardiac electrophysiology. Importantly, we show that the recording and analysis can be fully automated and the technology is readily available using commercial products.

[Analysis of mortality and its predictors in patients with contralateral hip fracture after femoral neck fracture]. / Ellenoldali csípotáji törésekkel összefüggo halálozás és kockázati tényezoinek vizsgálata.

INTRODUCTION: There is a high mortality with not well understood risk factors after the second hip fracture. AIM: Analysis of the 30- and 365-day mortality and its risk factors in patients with contralateral hip fracture. METHOD: Patients with contralateral hip fracture between 01 Jan 2000 and 31 Dec 2008 were identified among those who suffered their primary hip fracture in Hungary in 2000. Risk factors as age, sex, concomitant and chronic diseases, type of fracture and surgery, surgical complications, day of admission were analyzed by logistic and Cox regression as well as Kaplan-Meier analysis. RESULTS: There were 312 eligible patients identified with 8.3 % mortality rate at 30 and with 38,4% at 365 days respectively. Significant risk factors for the 30 day mortality were intertrochanteric type of fracture (OR: 4.722; HR: 4.129) and non operative management (OR: 7.357; HR: 6.317) while for the 365 day mortality those were older age (OR:1.070; HR:1.050) and type of surgery (OR: 0.450). CONCLUSION: Age, type of fracture and type of surgery proved to be risk factors. There is a need to identify further risk factors in order to develop an efficacious prevention strategy for the reduction of the mortality after the second hip fractures. Orv Hetil. 2017; 158(20): 783-790.

[Multidisciplinary approach of hip fractures based on Hungarian data]. / Csípotáji törések multidiszciplináris aspektusai magyarországi adatok alapján.

Hip fractures are described by increased mortality, loss of quality of life, functional decline and burden of diseases. They show a growing number worldwide. The aim of the present study is to summarise the existing data on the incidence, mortality, complications and rehabilitation of hip fractures, which relevance is reported only by few studies. To reduce mortality and complications of hip fractures the authors emphasize the importance of primary treatment within 12 hours, appropriate selection of surgical methods corresponding to the fracture type after the assessment of femoral head viability, vitamin D supplementation, same conditions for primary treatment during everyday of the week, and an adequate acute treatment and rehabilitation for patient's general health status. In the future integrated processing of multidisciplinary results of hip fractures based on Hungarian data can support the development of efficient treatment and prevention strategies, which can be advantageous for the patient, families, health care system, and the society, too, by the reduction of costly complications of hip fracture healing and mortality. Orv. Hetil., 2016, 157(37), 1469-1475.

[Impact of microRNAs on molecular epidemiology]. / Mikro-RNS-ek jelentosége a molekuláris epidemiológiában.

Cancer research concerning short non-coding RNA sequences and functionally linked to RNA interference (RNAi) have reached explosive breakthrough in the past decade. Molecular technology applies microRNA in extremely wide spectrum from molecular tumor prediction, diagnostics, progression monitoring and prevention. Functional analysis of tissue miRNA and cell-free serum miRNA in posttranscription and translation regulation innovated and restructured the knowledge on the field. This review focuses on molecular epidemiology and primary prevention aspects of the small non-coding RNA sequences.

Non-operative predictors for subsequent interventions after intracapsular femoral neck fractures in elderly: A comprehensive analysis of the Hungarian nationwide health insurance database.

OBJECTIVES: To investigate the correlation between non-operative prognostic factors and non-prosthetic fracture-related treatments following internal fixation of intracapsular femoral neck fractures in elderly patients. DESIGN AND SETTING: Retrospective observational cohort study. Comprehensive analysis of the Hungarian nationwide health insurance database. PARTICIPANTS: Data of in-patient records with S7200 ICD-10 codes were collected from the Hungarian National Health Insurance Fund Administration (HNHIFA) and from the health care provider institutes. The patients with femoral neck fractures in the year of 2000, following reduction and internal fixation aged 60 years or older were evaluated. The secondary, non-prosthetic fracture related treatments during the 8 year follow-up period were registered. MAIN OUTCOME MEASURES: Of the prognostic factors, age, gender, type of fracture, season and day of the primary surgery, length of waiting time to the operation and the accompanied diseases were evaluated as risk factors for all type of fracture-related further interventions, with the exception of arthroplasties. RESULTS: A total of 2895 patients with intracapsular femoral neck fractures met the study criteria. The mean age was 77.96 years (SD: 8.54). The cohort of the patients was observed for a total of 10,077.8 person-years. The non-prosthetic fracture related treatment was performed in 265 patients (9,2%); the median of the time elapsed to the secondary definitive treatment was 3.5 months. With Cox regression analysis, significant correlation was revealed between the incidence of non-prosthetic treatment and younger age (year, HR = 0.977, p = 0.002), surgical delay (12-24 h vs 0-6 h, HR = 1.518, p = 0.023; 24h+ vs 0-6 h, HR = 1.372, p = 0.050), season of primary osteosynthesis (fall vs summer, HR = 0.636, p = 0.012), and type of femoral neck fracture (intracapsular displaced vs intracapsular undisplaced, HR = 1.340, p = 0,047). There was no significant effect of the day of primary surgery, the gender and the presence of co-morbidities on the incidence of further surgical interventions. CONCLUSION: The summertime primary surgical intervention, delay of surgery longer than 12 h and type of femoral neck fracture are independent predictors of non-prosthetic further treatment of femoral neck fractures in elderly patients. LEVEL OF EVIDENCE: Level IV, evidence from cohort studies.

Integration of mechanical conditioning into a high throughput contractility assay for cardiac safety assessment.

INDUCTION: Despite increasing acceptance of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in safety pharmacology, controversy remains about the physiological relevance of existing in vitro models for their mechanical testing. We hypothesize that existing signs of immaturity of the cell models result from an improper mechanical environment. With the presented study, we aimed at validating the newly developed FLEXcyte96 technology with respect to physiological responses of hiPSC-CMs to pharmacological compounds with known inotropic and/or cardiotoxic effects. METHODS: hiPSC-CMs were cultured in a 96-well format on hyperelastic silicone membranes imitating their native mechanical environment. Cardiomyocyte contractility was measured contact-free by application of capacitive displacement sensing of the cell-membrane biohybrids. Acute effects of positive inotropic compounds with distinct mechanisms of action were examined. Additionally, cardiotoxic effects of tyrosine kinase inhibitors and anthracyclines were repetitively examined during repeated exposure to drug concentrations for up to 5 days. RESULTS: hiPSC-CMs grown on biomimetic membranes displayed increased contractility responses to isoproterenol, S-Bay K8644 and omecamtiv mecarbil without the need for additional stimulation. Tyrosine kinase inhibitor erlotinib, vandetanib, nilotinib, gefitinib, A-674563 as well as anthracycline idarubicin showed the expected cardiotoxic effects, including negative inotropy and induction of proarrhythmic events. DISCUSSION: We conclude that the FLEXcyte 96 system is a reliable high throughput tool for invitro cardiac contractility research, providing the user with data obtained under physiological conditions which resemble the native environment of human heart tissue. We showed that the results obtained for both acute and sub-chronic compound administration are consistent with the respective physiological responses in humans.

Cross - site comparison of excitation-contraction coupling using impedance and field potential recordings in hiPSC cardiomyocytes.

INTRODUCTION: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines. METHODS: Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed. RESULTS: Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values. DISCUSSION: With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

The opioid oxycodone use-dependently inhibits the cardiac sodium channel NaV 1.5.

BACKGROUND AND PURPOSE: Oxycodone is a potent semi-synthetic opioid that is commonly used for the treatment of severe acute and chronic pain. However, treatment with oxycodone can lead to cardiac electrical changes, such as long QT syndrome, potentially inducing sudden cardiac arrest. Here, we investigate whether the cardiac side effects of oxycodone can be explained by modulation of the cardiac Nav 1.5 sodium channel. EXPERIMENTAL APPROACH: Heterologously expressed human Nav 1.5, Nav 1.7 (HEK293 cells) or Nav 1.8 channels (mouse N1E-115 cells) were used for whole-cell patch-clamp electrophysiology. A variety of voltage-clamp protocols were used to test the effect of oxycodone on different channel gating modalities. Human stem cell-derived cardiomyocytes were used to measure the effect of oxycodone on cardiomyocyte beating. KEY RESULTS: Oxycodone inhibited Nav 1.5 channels, concentration and use-dependently, with an IC50 of 483 µM. In addition, oxycodone slows recovery of Nav 1.5 channels from fast inactivation and increases slow inactivation. At high concentrations, these effects lead to a reduced beat rate in cardiomyocytes and to arrhythmia. In contrast, no such effects could be observed on Nav 1.7 or Nav 1.8 channels. CONCLUSIONS AND IMPLICATIONS: Oxycodone leads to an accumulation of Nav 1.5 channels in inactivated states, with a slow time course. Although the concentrations needed to elicit cardiac arrhythmias in vitro are relatively high, some patients under long-term treatment with oxycodone as well as drug abusers and addicts might suffer from severe cardiac side effects induced by the slowly developing effects of oxycodone on Nav 1.5 channels.

Risk factors for contralateral hip fractures following femoral neck fractures in elderly: analysis of the Hungarian nationwide health insurance database.

OBJECTIVES: This study aims to investigate the significance of demographic and clinical factors on incidence of second (contralateral) hip fracture in elderly Hungarian population using the nationwide health insurance database in Hungary. PATIENTS AND METHODS: The study included a total of 3,783 patients (917 males, 2,866 females) treated for primary monotraumatic femoral neck fractures caused by low-energy trauma in the year 2000. Cox regression and Kaplan-Meier survival analyses, and log-rank test were performed to evaluate the following prognostic factors: age, gender, place of living, type of primary fracture and surgical intervention, hospital providing treatment for primary fracture, and comorbidities. RESULTS: A total of 312 patients (8.2%) suffered second hip fractures. The univariate Cox regression analysis showed a significantly higher risk for second hip fracture in patients having advanced age (p=0.001), female gender (p=0.022), living in capital (p=0.024), and having arthroplasty (p=0.001). Advanced age (p≤0.001) and having arthroplasty (p=0.004) were significant risk factors for second hip fractures according to multivariate analysis. Log-rank test showed significantly longer survival in females (p<0.001) than in males and in patients with arthroplasty (p=0.013) compared with those having osteosynthesis. CONCLUSION: Identification of high-risk groups for second hip fractures is needed to establish effective prevention strategies. Our study demonstrates that the risk of suffering from second hip fractures is higher in females, elderly population, those living in the capital, and patients having undergone arthroplasty.

Safety pharmacology studies using EFP and impedance.

INTRODUCTION: While extracellular field potential (EFP) recordings using multi-electrode arrays (MEAs) are a well-established technique for monitoring changes in cardiac and neuronal function, impedance is a relatively unexploited technology. The combination of EFP, impedance and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has important implications for safety pharmacology as functional information about contraction and field potentials can be gleaned from human cardiomyocytes in a beating monolayer. The main objectives of this study were to demonstrate, using a range of different compounds, that drug effects on contraction and electrophysiology can be detected using a beating monolayer of hiPSC-CMs on the CardioExcyte 96. METHODS: hiPSC-CMs were grown as a monolayer on NSP-96 plates for the CardioExcyte 96 (Nanion Technologies) and recordings were made in combined EFP and impedance mode at physiological temperature. The effect of the hERG blockers, E4031 and dofetilide, hERG trafficking inhibitor, pentamidine, ß-adrenergic receptor agonist, isoproterenol, and calcium channel blocker, nifedipine, was tested on the EFP and impedance signals. RESULTS: Combined impedance and EFP measurements were made from hiPSC-CMs using the CardioExcyte 96 (Nanion Technologies). E4031 and dofetilide, known to cause arrhythmia and Torsades de Pointes (TdP) in humans, decreased beat rate in impedance and EFP modes. Early afterdepolarization (EAD)-like events, an in vitro marker of TdP, could also be detected using this system. Isoproterenol and nifedipine caused an increase in beat rate. A long-term study (over 30h) of pentamidine, a hERG trafficking inhibitor, showed a concentration and time-dependent effect of pentamidine. DISCUSSION: In the light of the new Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative to improve guidelines and standardize assays and protocols, the use of EFP and impedance measurements from hiPSCs may become critical in determining the proarrhythmic risk of potential drug candidates. The combination of EFP offering information about cardiac electrophysiology, and impedance, providing information about contractility from the same area of a synchronously beating monolayer of human cardiomyocytes in a 96-well plate format has important implications for future cardiac safety testing.

Identification of Drug-Drug Interactions In Vitro: A Case Study Evaluating the Effects of Sofosbuvir and Amiodarone on hiPSC-Derived Cardiomyocytes.

Drug-drug interactions pose a difficult drug safety problem, given the increasing number of individuals taking multiple medications and the relative complexity of assessing the potential for interactions. For example, sofosbuvir-based drug treatments have significantly advanced care for hepatitis C virus-infected patients, yet recent reports suggest interactions with amiodarone may cause severe symptomatic bradycardia and thus limit an otherwise extremely effective treatment. Here, we evaluated the ability of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to recapitulate the interaction between sofosbuvir and amiodarone in vitro, and more generally assessed the feasibility of hiPSC-CMs as a model system for drug-drug interactions. Sofosbuvir alone had negligible effects on cardiomyocyte electrophysiology, whereas the sofosbuvir-amiodarone combination produced dose-dependent effects beyond that of amiodarone alone. By comparison, GS-331007, the primary circulating metabolite of sofosbuvir, had no effect alone or in combination with amiodarone. Further mechanistic studies revealed that the sofosbuvir-amiodarone combination disrupted intracellular calcium (Ca2+) handling and cellular electrophysiology at pharmacologically relevant concentrations, and mechanical activity at supra-pharmacological (30x Cmax) concentrations. These effects were independent of the common mechanisms of direct ion channel block and P-glycoprotein activity. These results support hiPSC-CMs as a comprehensive, yet scalable model system for the identification and evaluation of cardioactive pharmacodynamic drug-drug interactions.

Incubator proof miniaturized Holomonitor to in situ monitor cancer cells exposed to green tea polyphenol and preosteoblast cells adhering on nanostructured titanate surfaces: validity of the measured parameters and their corrections.

The in situ observation of cell movements and morphological parameters over longer periods of time under physiological conditions is critical in basic cell research and biomedical applications. The quantitative phase-contrast microscope applied in this study has a remarkably small size, therefore it can be placed directly into a humidified incubator. Here, we report on the successful application of this M4 Holomonitor to observe cancer cell motility, motility speed, and migration in the presence of the green tea polyphenol, epigallocatechin gallate, as well as to monitor the adhesion of preosteoblast cells on nanostructured titanate coatings, relevant for biomedical applications. A special mechanical stage was developed to position the sample into that range of the optical arrangement where digital autofocusing works with high reproducibility and precision. By in-depth analyzing the obtained single cell morphological parameters, we show that the limited vertical resolution of the optical setup results in underestimated single cell contact area and volume and overestimated single cell averaged thickness. We propose a simple model to correct the recorded data to obtain more precise single cell parameters. We compare the results with the kinetic data recorded by a surface sensitive optical biosensor, optical waveguide lightmode spectroscopy.

MicroRNA expressions in HPV-induced cervical dysplasia and cancer.

BACKGROUND/AIM: The role of oncogenic or high-risk human papillomavirus (HPV) in cervical carcinogenesis is inevitable, yet not fully understood. Detailed analysis of microRNA (miRNA) alterations occurring during high-risk HPV transformation will increase our current understanding over cervical carcinogenesis. The two main aims of the study were: (i) finding association between HPV infection characteristics and socio-demographic variables, (ii) finding an predictors of clinical outcome. MATERIALS AND METHODS: The expression levels of different microRNAs (miR-21, miR-27a, miR-34a, miR-155, miR-196a, miR-203) were determined in formalin-fixed paraffin-embedded (FFPE) human HPV-positive cervical dysplastic and tumorous tissue samples using quantitative real-time PCR (qPCR). Sociodemographic and life-style factors were also analyzed. RESULTS: The expression of miR-27a was significantly higher in cervical intraepithelial neoplasia (CIN)2-3 compared to CIN1 (p=0.023) and in squamous cell carcinoma (SCC) compared to CIN2-3 (p=0.033). Moreover, significantly lower levels of miR-34a were detected in CIN2-3 than in CIN1 (p=0.041) and in SCC than in CIN2-3 (p=0.021). Furthermore, we found significant differences in subjects with multiple HPV in miR-27a (p=0.015) and miR-203 (p=0.025) in CIN2-3 compared to CIN1 and miR-21 (p=0.002), mir-27a (p=0.001) and miR-34a (p=0.001) in SCC/CIN2-3. Expression of miR-27a, showing up-regulation in CIN2-3 compared to CIN1 (p=0.028) and miR-34a (down-regulated), correlated with HPV 16 positivity (CIN2-3/CIN1: p=0.027 and SCC/CIN2-3: p=0.036). MiR-34a expression was also significantly altered in connection to smoking status and presence of HPV 16. CONCLUSION: The demand for additional, alternative molecular biomarkers with prognostic potential is strong. Evaluation of miRNA expression might be helpful to distinguish different cervical lesions and might be able to help in the prediction of HPV infection outcome.

Alteration of miRNA expression correlates with lifestyle, social and environmental determinants in esophageal carcinoma.

BACKGROUND/AIM: Esophageal cancer (EC) is the eighth most common cancer with a highly aggressive potency. Considering the poor survival of esophageal carcinoma there is a need for useful molecular biomarkers for prevention and early detection. Our aim was to determine the significance of altered microRNA (miRNA) expression in esophageal cancer, in relation to lifestyle, social and environmental factors. MATERIALS AND METHODS: The relative expression levels of the following miRNAs: miR-21, miR-143, miR-196a, miR-203, miR-205 and miR-221 were monitored in control and esophageal squamous-cell carcinoma (ESCC) samples using real-time polymerase chain reaction (RT-PCR). miRNA expression pattern of tumor tissues were evaluated according to patients' social status, living condition, smoking and drinking habits alone and in combinations. RESULTS: miR-21, miR-143, miR-203, miR-205 and miR-221 were over-expressed in esophageal cancer compared with normal tissues. Increased expression of miR-205 was related to smoking, while excessive alcohol consumption showed a correlation with under-expression of miR-143, miR-203 and miR-205 in tumor samples. Significant associations were detected between reduced expression of miR-143, miR-203 and low social status, and combination of smoking and heavy drinking. CONCLUSION: Alterations of miRNA expression in ESCC can be correlated with the presence of common risk factors. The altered expression of certain miRNAs could be used as novel molecular markers of esophageal carcinoma.

Red sludge-induced mRNA and miRNA expression alterations in vital organs of CBA/Ca mice.

Several studies have investigated the ecological and genotoxic effects of the red mud accident in 2010 in Ajka, Hungary, but none was designed to reveal the early biological effects of red mud exposure at the level of early-responding gene expression. To address relevant questions, in the present study expression alterations of oncogenes (c-myc, K-ras), tumor suppressor genes (Bcl2, p53) and apoptosis-regulatory micro(mi) RNAs (miR-21, miR-27a, miR-93, miR-221) were analized 1, 3, 6 and 24 h after a single intraperitoneal injection of red mud to CBA/Ca mice. We observed changes in the expression of all investigated mRNAs, miR-21 and miR-221 in the liver, spleen, lung, kidney and lymph nodes of mice. An overexpression of the investigated genes was observed, but the level and the peak of the alteration differed according to examined tissue.

Effects of purified glycerol from biodiesel on miRNAs compared to the expression profile of selected mRNAs in Balb/c mice.

BACKGROUND: We investigated the effect of corn-derived biodiesel glycerol on microRNAs (miRNAs) and mRNAs, which play a central role in regulating cell survival, apoptosis and carcinogenesis. MATERIALS AND METHODS: Inbred Balb/c mice were treated with purified glycerol from biodiesel for 24 hours. After administration, we determined the expressions of miR-21, miR-27a, miR-34a, miR-93, miR-143, miR-146a, miR-148a, miR-155, miR-196a, miR-203, miR-205, miR-221 and nuclear factor kappa-light-chain enhancer of activated B-cells-1 (Nfκb1), mitogen-activated protein kinase-8 (Mapk8) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-ras) genes in the liver of mice. RESULTS: We found a parallel altered expression of miRNAs and mRNAs in animals consuming biodiesel glycerol that compared to control mice; these alterations reached significant levels only in few cases. CONCLUSION: Biodiesel glycerol presents no higher risk for carcinogenicity or toxicity.