scholarly journals Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

2021 ◽  
Author(s):  
Dirk Steinritz ◽  
Robin Lüling ◽  
Markus Siegert ◽  
Julia Herbert ◽  
Harald Mückter ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Dna Adducts ◽  
Sulfur Mustard ◽  
Ex Vivo ◽  
Immunomagnetic Separation ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Islamic State ◽  
Rat Skin

AbstractSulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

scholarly journals Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

2021 ◽  
Author(s):  
Dirk Steinritz ◽  
Robin Lüling ◽  
Markus Siegert ◽  
Harald Mückter ◽  
Tanja Popp ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Enzyme Activity ◽  
Sulfur Mustard ◽  
Critical Role ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Special Focus ◽  
Rabbit Muscle ◽  
Muscle Creatine Kinase

AbstractCreatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

In Vivo and ex Vivo Approaches to Studying the Biomechanical Properties of Healing Wounds in Rat Skin

2013 ◽  
Vol 135 (10) ◽  
Author(s):  
Clare Y. L. Chao ◽  
Gabriel Y. F. Ng ◽  
Kwok-Kuen Cheung ◽  
Yong-Ping Zheng ◽  
Li-Ke Wang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Ex Vivo ◽  
Normal Skin ◽  
Biomechanical Properties ◽  
Skin Wound ◽  
Sprague Dawley ◽  
Rat Skin ◽  
Mechanical Stiffness ◽  
Air Jet

An evaluation of wound mechanics is crucial in reflecting the wound healing status. The present study examined the biomechanical properties of healing rat skin wounds in vivo and ex vivo. Thirty male Sprague-Dawley rats, each with a 6 mm full-thickness circular punch biopsied wound at both posterior hind limbs were used. The mechanical stiffness at both the central and margins of the wound was measured repeatedly in five rats over the same wound sites to monitor the longitudinal changes over time of before wounding, and on days 0, 3, 7, 10, 14, and 21 after wounding in vivo by using an optical coherence tomography-based air-jet indentation system. Five rats were euthanized at each time point, and the biomechanical properties of the wound tissues were assessed ex vivo using a tensiometer. At the central wound bed region, the stiffness measured by the air-jet system increased significantly from day 0 (17.2%), peaked at day 7 (208.3%), and then decreased progressively until day 21 (40.2%) as compared with baseline prewounding status. The biomechanical parameters of the skin wound samples measured by the tensiometer showed a marked reduction upon wounding, then increased with time (all p < 0.05). On day 21, the ultimate tensile strength of the skin wound tissue approached 50% of the normal skin; while the stiffness of tissue recovered at a faster rate, reaching 97% of its prewounded state. Our results suggested that it took less time for healing wound tissues to recover their stiffness than their maximal strength in rat skin. The stiffness of wound tissues measured by air-jet could be an indicator for monitoring wound healing and contraction.

scholarly journals Studies to Elucidate the Mechanism of Cardio Protective and Hypotensive Activities of Anogeissus acuminata (Roxb. ex DC.) in Rodents

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3471
Author(s):  
Fatima Saqib ◽  
Muhammad Arif Aslam ◽  
Khizra Mujahid ◽  
Luigi Marceanu ◽  
Marius Moga ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Ex Vivo ◽  
Inflammatory Cells ◽  
Left Ventricular ◽  
Guanosine Monophosphate ◽  
Sprague Dawley ◽  
Positive Effects ◽  
Creatine Kinase Mb

Anogeissus acuminata (Roxb. ex DC.) is a folkloric medicinal plant in Asia; including Pakistan; used as a traditional remedy for cardiovascular disorders. This study was planned to establish a pharmacological basis for the trivial uses of Anogeissus acuminata in certain medical conditions related to cardiovascular systems and to explore the underlying mechanisms. Mechanistic studies suggested that crude extract of Anogeissus acuminata (Aa.Cr) produced in vitro cardio-relaxant and vasorelaxant effects in isolated paired atria and aorta coupled with in vivo decrease in blood pressure by invasive method; using pressure and force transducers connected to Power Lab Data Acquisition System. Moreover; Aa.Cr showed positive effects in left ventricular hypertrophy in Sprague Dawley rats observed hemodynamically by a decrease in cardiac cell size and fibrosis; along with absence of inflammatory cells; coupled with reduced levels of angiotensin converting enzyme (ACE) and renin concentration along with increased concentrations of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP). In Acute Myocardial Infarction (AMI) model; creatine kinase (CK), creatine kinase-MB (CK-MB) and lactic acid dehydrogenase (LDH levels) were found to be decreased; along with decreased necrosis; edema and recruitment of inflammatory cells histologically. In vivo and ex vivo studies of Anogeissus acuminata provided evidence of vasorelaxant; hypotensive and cardioprotective properties facilitated through blockage of voltage-gated Ca++ ion channel; validating its use in cardiovascular diseases

Validation of an In vitro-in vivo Assay System for Evaluation of Transdermal Delivery of Caffeine

2019 ◽  
Vol 9 (1) ◽  
pp. 15-20 ◽  
Author(s):  
Fanni Farner ◽  
Luca Bors ◽  
Ágnes Bajza ◽  
Gellért Karvaly ◽  
István Antal ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Assay System ◽  
Diffusion Cell ◽  
Rat Skin ◽  
Topical Formulation ◽  
Franz Diffusion Cell ◽  
Topical Drugs ◽  
In Vivo Assay

Introduction: Degree of skin penetration of topical drugs and cosmetics is a crucial point concerning their effects and tolerability. For testing drug delivery across the dermal barrier different in vitro and in vivo assays have been developed. Caffeine has been shown to have beneficial effects against skin aging, sunburn and hair-loss, and it is protective against melanoma and non-melanoma type skin cancers. Aim of our study was to set up an assay system to evaluate caffeine penetration from topical formulation into the skin. </P><P> Methods: Franz diffusion cells consisting of either a filter paper or an artificial membrane or rat skin were used as in vitro/ex vivo test systems and transdermal microdialysis in anaesthetized rats was performed as an in vivo assay. </P><P> Results: Results indicate that Franz diffusion cell studies provide a good approximation of the release of caffeine from the formulation but are not able to differentiate between 2% and 4% cream concentrations. The maximum concentrations (Cmax) in case of the 2% cream formulation were 708.3 (2.7 μm pore), 78.7 (0.8 &#181;m pore), 45.3 (0.45 &#181;m pore) and 44.9 (rat skin) &#181;g/7.5 mL, respectively. The in vivo microdialysis experiments were in accordance with the in vitro and ex vivo results and gave more information on the dynamics and follicular and transcellular phases of drug penetration through the layers of the skin. </P><P> Discussion and Conclusion: Taken together, Franz diffusion cell and transdermal microdialysis are a good combination to evaluate caffeine release and penetration into the skin from the formulations tested. This system might also be used for rapid testing of other hydrophilic topical drugs and has a benefit in the prediction for human skin absorption and tolerability studies, in an early phase of drug development.

ALTERATIONS IN AUTOFLUORESCENCE SIGNAL FROM RAT SKIN EX VIVO UNDER OPTICAL IMMERSION CLEARING

2010 ◽  
Vol 03 (03) ◽  
pp. 147-152 ◽  
Author(s):  
E. V. MIGACHEVA ◽  
A. B. PRAVDIN ◽  
V. V. TUCHIN
Keyword(s):  
Fluorescence Spectroscopy ◽  
Ex Vivo ◽  
Rat Skin ◽  
Optical Clearing ◽  
Spectral Curves ◽  
The Common ◽  
First Time ◽  
Tissue Optical Clearing ◽  
Optical Immersion

For the first time, the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing. The glucose, glycerol and propylene glycol solutions were used as clearing agents. The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers. In this contribution, the common properties of autofluorescence variation during optical immersion clearing were determined. The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail. However, the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid. The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.

scholarly journals Alkylated albumin-derived dipeptide C(-HETE)P derivatized by propionic anhydride as a biomarker for the verification of poisoning with sulfur mustard

2021 ◽  
Author(s):  
Annika Richter ◽  
Markus Siegert ◽  
Horst Thiermann ◽  
Harald John
Keyword(s):  
Sulfur Mustard ◽  
Limit Of Detection ◽  
Thiol Group ◽  
Chemical Warfare Agent ◽  
Cysteine Residue ◽  
Selected Reaction Monitoring ◽  
Delayed Wound Healing ◽  
Propionic Anhydride ◽  
Product Ions

AbstractSulfur mustard (SM) is a banned chemical warfare agent recently used in the Syrian Arab Republic conflict causing erythema and blisters characterized by complicated and delayed wound healing. For medical and legal reasons, the proof of exposure to SM is of high toxicological and forensic relevance. SM reacts with endogenous human serum albumin (HSA adducts) alkylating the thiol group of the cysteine residue C34, thus causing the addition of the hydroxyethylthioethyl (HETE) moiety. Following proteolysis with pronase, the biomarker dipeptide C(-HETE)P is produced. To expand the possibilities for verification of exposure, we herein introduce a novel biomarker produced from that alkylated dipeptide by derivatization with propionic anhydride inducing the selective propionylation of the N-terminus yielding PA-C(-HETE)P. Quantitative derivatization is carried out at room temperature in aqueous buffer within 10 s. The biomarker was found to be stable in the autosampler at 15 °C for at least 24 h, thus documenting its suitability even for larger sets of samples. Selective and sensitive detection is done by micro liquid chromatography-electrospray ionization tandem-mass spectrometry (μLC-ESI MS/MS) operating in the selected reaction monitoring (SRM) mode detecting product ions of the single protonated PA-C(-HETE)P (m/z 379.1) at m/z 116.1, m/z 137.0, and m/z 105.0. The lower limit of detection corresponds to 32 nM SM in plasma in vitro and the limit of identification to 160 nM. The applicability to real exposure scenarios was proven by analyzing samples from the Middle East confirming poisoning with SM. Graphical abstract

scholarly journals Pathophysiology and inflammatory biomarkers of sulfur mustard-induced corneal injury in rabbits

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258503
Author(s):  
Dinesh G. Goswami ◽  
Neha Mishra ◽  
Rama Kant ◽  
Chapla Agarwal ◽  
Claire R. Croutch ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Sulfur Mustard ◽  
Corneal Thickness ◽  
Inflammatory Cells ◽  
Chemical Warfare Agent ◽  
Therapeutic Interventions ◽  
Dependent Manner ◽  
Corneal Injury ◽  
Cox 2

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions.

Kinetics of Rat Skin Optical Clearing at Topical Application of 40%Glucose: Ex Vivo and In Vivo Studies

2019 ◽  
Vol 25 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Daria K. Tuchina ◽  
Polina A. Timoshina ◽  
Valery V. Tuchin ◽  
Alexey N. Bashkatov ◽  
Elina A. Genina
Keyword(s):  
Topical Application ◽  
Ex Vivo ◽  
Rat Skin ◽  
Optical Clearing ◽  
Kinetics Of

scholarly journals In Vitro, Ex Vivo, Penetration (EpiDermTM) and in Vivo Dermatokinetics of Ketoconazole Loaded Solid Lipid Nanoparticles for Topical Delivery

2021 ◽  
Author(s):  
Mohhammad Ramzan ◽  
Samuel Gourion-Arsiquaud ◽  
Afzal Hussain ◽  
Jaspreet Singh Gulati ◽  
Qihong Zhang ◽  
...  
Keyword(s):  
Solid Lipid Nanoparticles ◽  
Ex Vivo ◽  
Lipid Nanoparticles ◽  
Laser Scanning Microscopy ◽  
Pharmacokinetic Parameters ◽  
Rat Skin ◽  
Solid Lipid ◽  
Ex Vivo Permeation

Abstract The study focused to optimize, evaluate and investigate mechanistic perspective of ketoconazole (KTZ) loaded solid lipid nanoparticles (KTZ-SLNs) for enhanced permeation across rat skin. KTZ-SLNs were evaluated for size, distribution, zeta potential (ZP), percent entrapment efficiency (%EE), drug release, morphology, thermal behavior (DSC), compatibility (FTIR) and solid state characterization (X-ray diffraction, XRD). Moreover, ex-vivo permeation and drug deposition into rat skin were conducted using Franz diffusion cell. Mechanistic evaluations were confirmed using confocal laser scanning microscopy and vibrational ATR methods using EpiDermTM model. An in vivo dermatokinetics study was performed to ensure KTZ access to the dermal region. Accelerated and photostability studies were conducted at different temperatures (0, 30, and 40 °C) for 12 months. The spherical optimized KOF1 showed optimal particle size (291 nm), and high negative ZP (-27.7 mV). Results of FTIR, DSC, and XRD confirmed compatibility of KTZ with excipients, purity of KTZ & dissolved KTZ in lipid matrix, and amorphous nature of KTZ-SLNs. In-vitro release was found to be slow and sustained whereas ex vivo permeation parameters were significantly high in KTZ-SLNs as compared to drug suspension and marketed product. Drug retention was 10- and -5 fold higher than KTZ-SUS and marketed product, respectively. Pharmacokinetic parameters were improved by SLNs formulation. Confocal raman spectroscopy experiment showed that KTZ-SLNs could penetrate beyond the human stratum corneum into viable epidermis. Fluorescent microscopy confirmed improved penetration of KTZ-SLNs was through human follicular pathway. KTZ-SLNs stable over 12 months under set conditions.

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