# Presentation: 621201

## BRITTON CHANCE

**B****RITTON ****C****HANCE**

**DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS and RADIOLOGY****UNIVERSITY OF PENNSLYVANIA****chance@mail.med.upenn.edu**

## Science in the 1930’s

**Discussion Topic: Was there communication from the Warburg, Meyerhoff, Embden glycolysis through Krebs’s citric acid cycle through Keilin’s cytochromes and is neurophysiology any better?**

## Mitochondrion

***Mitochondrion***

## Mitochondrial Free Radical Generation

- H2O2 Production

## Discovery of mitochondrial H2O2 generation

- Kinetics of H2O2 production by pigeon heart mitochondria in the presence of succinate and antimycin A.

***Discovery of mitochondrial H******2******O******2****** generation***

## Balance between the rate of uric acid oxidation and extra H2O2 production rate in perfused rat liver

***Balance between the rate of uric acid oxidation and extra H******2******O******2****** production rate in perfused rat liver***

## Proposed mechanism of the reactions of catalase compound I

***Proposed mechanism of the reactions of catalase compound I***

## Rates of H2O2 Production in Intact Liver

| Addition | H2O2 Production 
(nmol/min per gram liver wet wt) |
| --- | --- |
| None | 49 |
| Urate, 1 mM | 750 |
| Glycolate, 3 mM | 490 |
| Octanoate, 0.3 mM | 170 |
| Oleate, 0.1 mM | 66 |
| Decanoate, 0.085 mM | 151 |

***Rates of H******2******O******2****** Production in Intact Liver***

## Chemiluminescence of the perfused rat liver due to free radical generation

- tBuOOH and CumOOH were infused at the rates indicated by the numbers in the blocks. For the traces in the main figure and in the Inset, redsensitive EMI 9658 and green-sensitive RCA 8850 phototubes were used, respectively.

***Chemiluminescence of the perfused rat liver due to free radical generation***

## Fatty acid caused H2O2 generation

***Fatty acid caused H******2******O******2****** generation***

## H2O2 generation by myelocytes & polymorphonucleorleucocytes

- H2O2 production by both intact M and PML in the presence and in the absence of glucose. Both kinds of intact cells (lung M and PML) were suspended in Ca2+-free Krebs Ringer phosphate buffer, pH 7.4 in the presence or in the absence of 0.5 mM glucose. 1.0 M cytochrome c peroxidase was added to the cell suspension. The H2O2 production was recorded at 37oC after addition of opsonized zymozan.  ● + glucose; ○ – glucose.

***H******2******O******2****** generation by myelocytes & polymorphonucleorleucocytes***

## Free radical reaction mechanisms

- Stages of lipid peroxidation. The first and second stage are the classical reactions for formation and accumulation of lipoperoxides. The third stage, the breakdown of lipoperoxides, focuses particularly on radical reactions leading either to chemiluminescence or to the formation of penthane or ethane. Vitamin E (Vit. EH) stops the chain reaction of lipid peroxidation by reacting with lipid peroxy radicals (ROO·). The numbers in brackets express the estimated steady state concentrations or formation rates of oxygen metabolites. G-Per, glutathione peroxidase; Cat, catalase; SOD, superoxide dismutase; ROOH, lipid hydroperoxide; RO·, alkoxy radicals; HO·, hydroxyl radicals; O2-, superoxide anion; 1O2 ME, singlet oxygen monomol emission; 1O2, DE, singlet oxygen dimol emission.

***Free radical reaction mechanisms***

## Mitochondrial Bioluminescence

## Chemiluminescence detector

***Chemiluminescence detector***

## Chemiluminescence of rat organs

- Relationship between low-level chemiluminescence and content of endogenous peroxides in organs and tissues form normal animals and tumor-bearing animals. 1. Rat skeletal muscles (hindlimbs); 2. mouse sarcoma 180 (days 8-18); 3. Walker rat carcinoma (days 6-20); 4. rat brain (normal); 5. mouse omentum (normal); 6. rat liver (normal); 7. mouse brain with Ehrlich carcinoma (day 11); 8. liver of rat with Walker carcinoma (day 10); 9, 10. brain and liver of mouse with sarcoma 180 (day 18); 11, 12, liver and brain of mouse with sarcoma 180 (day 38).

***Chemiluminescence of rat organs***

## Chemiluminescence of cytochrome c

- Dependence of chemiluminescence on (a) cytochrome c concentration and (b) the [t-butyl hydroperoxide]/[ferricytochrome c] ratio. (a) Assay was carried out in the present of 5mM-t-butyl hydroperoxide. Different ratios shown in (b) were obtained by varying the hydroperoxide concentration and keeping ferricytochrome c concentration constant at 30 M.

***Chemiluminescence of cytochrome c***

## Respiratory Components and the Targets for Inhibitors

## Cover picture for J. Bioenergetics and Biomembranes

## Mitochondrial Redox Imaging

## Partial separation of the spectra of components of respiratory chain of rat liver mitochondria by various chemical treatments. The difference spectra were recorded by the dual wavelength spectrophotometer which plots the difference between the absorption of the reference sample of mitochondria (2 mg. of protein per cc.) and the experimental one.  In the trace, the difference is caused by anaerobiosis, which gives the oxidized minus reduced difference spectrum of mitochondria. The absorption peaks reading from right to left are caused by the reduction of cytochromes aa, ca, (aa) g,  and cg, and DPN. The trough at 465 mμ is caused by the reduction of oxidized flavoprotein.

**NADH **(40x)

**Flavoprotein **(5x)

*BC-X 156a*

## Isolated Mitochondrial Fluorescence Spectra

- State 3 - Active

- State 2 - Starved

- Chance and Baltscheffsky (1958)J. Biol. Chem. 233:3 736-739
*BC-X 155*

- 366 nm excitation

- 436 nm excitation

***Isolated Mitochondrial Fluorescence Spectra***

- State 4 - Resting

- State 3 - Active

- State 4 - Resting

- Flavoprotein

- NADH

- Chance and Schoener (1966) J. Biol. Chem. 241:20 4567-4573
*BC-X 167*

## A Further Increase of Sensitivity/Specificity in the NIR Region

***1)  Intrinsic beacons:****** ******NADH, Fp ***
***2)  Blood Pooling:****** ******ICG***
***Affinity Labels:******	******Somatostatin ******	******Bombesin ******	******LDL Folate ******	******Fluorescent Glucose ******	******Integrin***
***4)  Stealth Beacons:******	******Cathepsin B***
***5)  Genetic Expression: ******GFP***
***Quantum Dots***
***NIR-Emissive Polymersomes***

## GFP expression K1735 Murine Melanoma in mouse gives very strong fluorescence at 520 nm when excited at 436 nm

**surface imaging GFP expression**

*Courtesy W. El-Deiry*

- Surface signal only (~ 1mm deep)

***GFP expression K1735 Murine Melanoma in mouse gives very strong fluorescence at 520 nm when excited at 436 nm***

## Time-Domain Imaging and Reconstruction (Slices)

- 24 h

- Slices (1, *z *= 0 mm
- 20, *z *= 19 mm)

*Courtesy W. Baker*

***Time-Domain Imaging and Reconstruction (Slices)***

## Metabolitic Histopathology: High Resolution Cryo-imaging

- Chance and Quistorff (1985) Anal. Biochem. 148: 389-400
- “Snap shot” of metabolism at the freezing moment
- Higher fluorescence quantum yield (5-10 fold) at liquid N2 temperature; thus, better S/N ratio
- Allows high level of details and signal averaging
- Allows 3D imaging

## Redox Images of HepG2 Tumor with DiR-LDL Probe

**NADH**

**DiR-LDL**

** ****1 **

**0.5 **

**  ****0 **

**Size: 1cm x 1cm**

***Redox Images of HepG2 Tumor with DiR-LDL Probe***

## Cy5.5 in HT 1080  with 2 nmol PGC-Cy5.5

- Cy5.5

- Redox Ratio

- NADH

- Fp

**Ex: 366nm**
**Em: 455nm**

**Ex: 670nm**
**Em: 695nm**

**Ex: 436nm**
**Em: 525nm**

**NADH/(NADH+Fp)**

***Cy5.5 in HT 1080  with 2 nmol PGC-Cy5.5***

*LLZ-2*

- Identifying tumor with stealth beacon Cy5.5 (red and green)with reduced redox ratio (red).

- 1 cm x 1 cmDepth: 800 μm

## Fluorescent Glucose Structures

- Pyro-2DG

- BChl-2DG

- 2-NBDG

## C-MYC tumor in mouse200l 0.5mg/ml Pyro-glucose after 2.5hrs

***C-MYC tumor in mouse******200************l 0.5mg/ml Pyro-glucose after 2.5hrs***

**FP**

**NADH**

**R2 (Pyro-glucose)**

**Ratio [PN/(FP+PN)]**

**Hb**

**T-Hb**

**Histogram****of reduced ratio**

- 02111301

- 02111302

*ZZh 17a*

**Depth: 700 mm**

***(11/06/02) #M1***

**Histogram****of pyro-glucose**

**Scan Area: 1.024 x 1.024 cm****2**

**Ratio [FP/(FP+PN)]**

**Histogram****of oxidized ratio**

**Note congruence of mitochondrial NADH and Pyro-glucose**

## NADH VS Pyro-Glucose

## Evidence of Pyro-2DG’s Internalization via the GLUT Pathway

- Inhibition of 15M Pyro-2DG by 250M phloretin and 20M cytochalasin B (GLUT inhibitors) in HepG2 cells.

**250**

**200**

**150**

**100**

**50**

**0**

**250**

**200**

**150**

**100**

**50**

**0**

## Mitochondrial Flavoprotein and NADH

***Apoptosis Signals***

## ?

*Courtesy R. Garrett and C. Grisham*

***Hypothetical Sites for Apoptosis***

## Prostate Tumor SK3R Treated with Taxol 40 g/mouse

**Depth: 400****μm   Dimension: 1cm × 1cm**

***Prostate Tumor SK3R Treated with Taxol 40 ************g/mouse***

- BZ17_2,04082401

- FP

- NADH

- FP/(FP+NADH)

## PDT Protocol for Pyro-2DG

**Light: 670 nm laser; 75mW/cm****2****; 33 min; 150 J/cm****2****;**
**	****Drug: 0.5 mg/mouse; Tumor size: 1 cm diameter (10 days)**
**Administration: bolus tail vein injection (6 nmol/kg)**
**PDT: 2 hours post injection; Frozen 15 min after light treatment; Anesthesia: continuous**

## New PDT Concept Based on 1O2 Quenching and Activation

*J. Chen, K. Stefflova, et al. JACS 2004, 126: 11450-11451*

**Protease-triggered PDT beacon**

## Photodynamic Therapy (PDT)

***Intensity: 0.59mW/cm******2****** (total dose = 150J/cm******2******)***

***Photodynamic Therapy (PDT)***

**Control**

**PDT**

## Mitochondrial redox response to PDT of Pyropheophorbide 2-deoxyglucosamide (Pyro-2DG)

- 04041302, 04041400, 04041402 (600, 700, 900μm)

**HepG2 tumor in** **SKID mouse model (tumor: ~1cm diameter) **
**Cryo-image 50μm resolution (x,y) 100μm resolution (z)**
**Pyro-2DG: ****5mg/kg**
**PDT (****spot, 1mm diameter****) intensity: 0.59mW/cm****2**** (total dose = 150J/cm****2****)**

**Redox State: Fp/(Fp+NADH) (%)**

***reduced***

**Irradiated**

**Non-Irradiated**

**Total ~ 3 x 10****4**** voxels**

***oxidized***

_**600-900μm Deep**_

_**300-500μm Deep**_

**Numbers of Observation**

**Redox State: Fp/(Fp+NADH) (%)**

***reduced***

***oxidized***

- 04041200, 04041300, 04041301 (300, 400, 500μm)

- BZ11 1T

**Total ~ 3 x 10****4**** voxels**

***Mitochondrial redox response to PDT of Pyropheophorbide 2-deoxyglucosamide (Pyro-2DG)***

## PDT (Whole Field)

***Intensity: 151mW/cm******2****** (total dose = 75J/cm******2******)***

**Depth: 800μm     Dimension: 1 cm ****×**** 1 cm**

**Fp**

**NADH**

**Fp/(Fp+NADH)**

**Pyro-2DG**

***PDT (Whole Field)***

## Mitochondrial redox response to PDT of Pyropheophorbide 2-deoxyglucosamide (Pyro-2DG)

_**800-1000μm Deep**_

_**800-1000μm Deep**_

**Redox State: Fp/(Fp+NADH) (%)**

***reduced***

***oxidized***

**Numbers of Observation**

**Redox State: Fp/(Fp+NADH) (%)**

***reduced***

***oxidized***

- 04042202, 04042600, 04042601 (800, 900, 1000μm)     04041600, 04041601, 04041602 (800, 900, 1000μm)

| HepG2 tumor in SKID mouse model (tumor: ~1cm diameter) 
Cryo-image 50μm resolution (x,y) 100μm resolution (z)
Pyro-2DG: none
PDT (whole field) intensity: 151mW/cm2 (total dose = 250J/cm2) | HepG2 tumor in SKID mouse model (tumor: ~1cm diameter) 
Cryo-image 50μm resolution (x,y) 100μm resolution (z)
Pyro-2DG: 5mg/kg
PDT (whole field) intensity: 151mW/cm2 (total dose = 75J/cm2) |
| --- | --- |

- BZ11 6T, 2T

**Total ~ 3 x 10****4**** voxels**

**Total ~ 3 x 10****4**** voxels**

**Non-Irradiated**

**Irradiated**

***Mitochondrial redox response to PDT of Pyropheophorbide 2-deoxyglucosamide (Pyro-2DG)***

## Two Photon Imaging of NADH and Fp in Cancers

## Automated Cryo Two-Photon Microscopy

**Faster scan rate**
**Better SNR**
**3D, Very high resolution images of large tissue samples**

***Automated Cryo Two-Photon Microscopy***

## One photon  NADH, area of view 7mm 128128 pixels

- One photon  NADH, area of view 7mm 128128 pixels

- Two photon NADH, excitation 740nm, 80mW area of view 1000 m, 512512 pixels

- One photon Flavoprotein, area of view 7mm 128128 pixels

- Two photon Flavoprotein, excitation 800nm 80mW, area of view 1000 m, 512512 pixels

## Histogram Displays of Heterogeneity of Cancer 

## One photon  NADH, area of view 7mm 128128 pixels

- One photon  NADH, area of view 7mm 128128 pixels

- Two photon NADH, excitation 740nm, 80mW area of view 1000 m, 512512 pixels

- One photon Flavoprotein, area of view 7mm 128128 pixels

- Two photon Flavoprotein, excitation 800nm 80mW, area of view 1000 m, 512512 pixels

##  Quantum Dots

***The Future:******Especially Bright Fluorochromes***

## Quantum Dots

**Excitation (blue) and Emission (orange) spectra for Qdot 605 streptavidin conjugates.**

*Quantum Dot Corporation, March 2003*

***Quantum Dots***

**Emission spectra showing narrow, symmetric peaks of various Qdot nanocrystals.**

## Slide 48

## Summary on molecular beacons

## Collaborators -  University of Pennsylvania

***Tao Ao******Baruch Ben-Dor******Ping Huang******Huamin Wang******Gwen Lech ******Samantha Gaw******Emily Hwang******Connie Y. Li******Zhongyao Zhao Bleu Zhong******Jun Zhang ******Juan Du ******Xiaohong Li ******Sarat Pothuri*** ***Walid M. Fahssi***

**Center for Biomedical Optics**

**Physics Dept.**

***Arjun Yodh***

**Radiology Dept.**

***J.S. Leigh******Mitchell Schnall******Jerry Glickson Gang Zheng ***

**Neurology Dept.**

***William Bank***

**Chemistry Dept.**

***Al Gerwitz******Ponzy Lu***

**Children’s Hospital**

***Tony Simon***

***Lanlan Zhou******Cindy Wang ******Gunay Yurtsever******Sammy Shan******Ye Xing******Barry John-Chuan David R. Busch Mahsa Ranji******Joohee Im ******Dan Huang     Yingting Liu******Xiang Cao ******Hui Sun ******Ulas Sunar ******Chilton Alter***

**NTROI**

***Wafik S. El-Deiry***

## Collaborators -  Elsewhere

***Bruce Tromberg***U.C. Irvine, CA

***Nimmi Ramanujam***Univ. of Wisconsin, Madison, WI

***John Schotland***Univ. of Pennsylvania

***Michael Patterson***Hamilton, Ontario, Canada

***Eva Sevick-Muraca***Purdue Univ. West Lafayette, IN

***Hanli Liu***Univ. of Texas, Arlington, TX

***Ken Rundell***Olympic Center, Lace Placid, NY

***Sam Achilefu***Mallinckrodt Labs, St. Louis, MO

***Maria Delivoria******Enla Anday***Medical College of PAPhiladelphia, PA

***Ralph Weissleder ***MGH, Boston, MA

***Susan Briest***Leipzig, Germany

***Kevin McCully***Univ. of GeorgiaAthens, GA

***Takafumi Hamaoka***Toyko, Japan

***H. Koizumi***Hitachi, Tokyo, Japan

***Maria Anglina ******Franchescini***MGH, Boston, MA

***Christopher H. Contag***NTROI, Stanford

***Ching H. Tung*** MGH, Boston, MA