The Genetically-encoded Photosensitizer mini Singlet Oxygen Generator produces superoxide

together, we show that MC exposure exacerbates NAFLD

Reaction Mechanisms and

pathology via NOX2 dependent peroxynitrite generation by increasing miR21 levels.

Methodologies/

DOI: 10.1016/j.freeradbiomed.2017.10.083

Other Redox-based Processes 70

71

Microcystin exposure Exacerbates Non-

The Genetically-encoded Photosensitizer

alcoholic Fatty Liver Disease (NAFLD) via

mini Singlet Oxygen Generator produces

NOX2 Dependent Activation of miR21-

superoxide

induced Inflammatory Pathways Muayad Albadrani1, Firas Alhasson1, Diptadip Dattaroy1, Varun Chandrashekaran1, Ratanesh Seth1, Mitzi Nagarkatti2, Prakash Nagarkatti2, and Saurabh Chatterjee1 University of South Carolina, USA

2

University of South Carolina-School of Medicine, USA

15%

of

the

cases

harmful role within a cell, ROS can also have a protective

progressing

to

steatohepatitis or cirrhosis and eventually to hepatocellular carcinoma. Though significant progress has been made in unravelling the pathophysiology of the disease, the effect of environmental and genetic factors in an underlying NAFLD pathophysiology remains unclear. The present report tests the

hypothesis

that

microcystin

(MC),

a

principal

component of harmful algal blooms activates Kupffer cells and hepatic stellate cells, crucial mediators in hepatic fibrogenesis via NOX2 dependent pathway in an underlying condition of NAFLD.

Mice that had mild steatosis and

showed signs of NAFLD were exposed to microcystin via an intraperitoneal route. Results showed that MC exposure led to exacerbation of NAFLD pathology with increased CD68 immunoreactivity, release of proinflammatory cytokines and stellate cell activation, a process that was attenuated in miR21 knockout mice and mice that lacked the p47 phox gene. Mechanistically, immortalized Kupffer cells treated with apocynin or nitrone spin trap DMPO had significantly decreased CD68, decreased miR21 and α-SMA levels signifying the role of NOX2-dedendent reactive oxygen species in miR21-induced Kupffer cell activation and stellate cell pathology. Further, NOX2 dependent peroxynitrite generation was primarily responsible for cellular events observed following MC exposure since incubation with phenyl boronic acid attenuated miR21 levels, Kuppfer cell activation

and

inflammatory

cytokine

release.

University of Rochester, USA

While reactive oxygen species (ROS) are known to have a

Non-alcoholic fatty liver disease is an emerging global with

Andrew Wojtovich1 1

1

pandemic

Miriam Barnett1, Timothy Baran1, Thomas Foster1, and

Taken

signaling role. One limitation to differentiating these roles is the inability to spatially and temporally control ROS production. ROS-generating proteins (RGPs) overcome this limitation by using light to control ROS production. RGPs have been used in a variety of applications, from cell ablation to mutagenesis; however, the type of ROS produced is often not well characterized. One RGP, mini “Singlet Oxygen Generator” was reported to generate large quantities of singlet oxygen. However, the contribution of superoxide remains unclear due to indirect measures with confounding interpretations.

We

measured

the

light-dependent

superoxide production of purified miniSOG using HPLC separation of dihydroethidium oxidation products. We demonstrate that miniSOG generates superoxide in an SODdependent

and

azide/catalase-independent

manner.

miniSOG’s superoxide production rate was lower than its free

chromophore,

produced

superoxide

flavin at

mononucleotide. a

rate

of

miniSOG

~4.0

µmol

superoxide/min/µmol photosensitizer for an excitation fluence rate of 5.9 mW/mm2 at 470±20 nm, and the rate remained

consistent

across

fluences.

This

contrasts

miniSOG’s singlet oxygen production which has been shown to be fluence-dependent. Understanding the type and amount of biologically available ROS produced by miniSOG can improve the experimental design and interpretation of the results. Our results may aid in the design of new RGPs that can be more selective in their photosensitization mechanism.

SfRBM 2017

61

73

DOI: 10.1016/j.freeradbiomed.2017.10.084

Chemical Trapping and Characterization of small Oxoacids of Sulfur (SOS) and

72

Selenium (SOSe) Generated in Aqueous Oxidations of H2S and H2Se

A Sensitive and Cost-effective

Murugaeson Ravi Kumar1 and Patrick Farmer1

Fluorescence-based Pyrogallol red assay for H2O2: Applied to Detect H2O2 Mobility Between Culture Wells

Gwenny Z. Go and Aimee Eggler 1

1

1

Baylor University, USA

Small oxoacids of sulfur (SOS) are elusive molecules like 1

[H2O2]

Villanova University, USA

[ROOH]

A novel fluorescence-based assay to quantitate hydrogen

[HOCl] H2S

peroxide levels in biological samples was developed with red (PGR) and horseradish peroxidase (HRP). These

H2S

characteristics of PGR, as well as its ability to be readily oxidized by various species and its absorbance maxima at

to the low μM range. Given its structural similarity to other anionic xanthene dyes, such as fluorescein, we hypothesized PGR would also have fluorescent properties, which might extend its linear range of H2O2 detection. We find that the

reduced and oxidized forms of PGR both fluoresce, with distinct excitation and emission spectra. The linear range of H2O2 detection is significantly extended by fluorescence

detection. A rapid 96-well plate assay was developed. Controls are included for 1) specifically detecting H2O2 by

subtracting out any changes observed after treating samples with catalase and 2) accounting for any nucleophilic

and

sulfinic

acid,

generated

by

oxidation

of

hydro-gen sulfide, H2S,

H2S

in

aqueous

solution. Like their

[Cbl] [MP-11]

of assays to quantitate levels of small molecule oxidants and

the linear range of H2O2 detection by this method is limited

H2S

[Mb]

540 nm at physiological pH, have led to its use in a number

catalyzed, such as by HRP, H2O2 oxidized PGR. We find that

H2S

acid,

HSOH,

HS(O)OH,

HSOnH

the relatively inexpensive and non-toxic reagents pyrogallol

antioxidants, metal ions, and catalase. When the reaction is

H2S

sulfenic

alkyl

homologs,

SOS may exhibit both nucleophilic

and electrophilic reactivity due to interconversion between S(II) and S(IV) tautomers. We find that SOS may be trapped in situ by derivate-ization with nucleophilic and electrophilic trapping agents and then characterized by high resolution LC/MS. Herein, we compare SOS formation from H2S oxidation by a variety of biologically relevant oxidants.

Analogous small oxoacids of selenium, SOSe, are generated during aqueous oxidations of hydrogen selenide (H2Se). These SOS and SOSe species appear relatively long lived in

aqueous solution, and thus may be involved in the observed physiological effects derived from H2S and potentially H2Se. DOI: 10.1016/j.freeradbiomed.2017.10.086

quenching of H2O2 (such as by glutathione) by pretreatment

with the electrophile N-ethylmaleimide. This assay was then utilized to determine whether and to what extent H2O2

accumulated in neighboring wells in a multi-well cell culture plate, due to volatilization from a treated well. We find that H2O2 does travel from a treated well to a

neighboring well.

DOI: 10.1016/j.freeradbiomed.2017.10.085

74 Determination of Vitamin D in Tears of Healthy Individuals by the Electrochemiluminescent Method Renato Leca1, Lai Yu Tsun1, and Fernando Fonseca1 1

62

Faculdade de Medicina do ABC, Brazil

SfRBM 2017