Michael Owens

Trail of breadcrumbs

Discovering the molecular mechanisms of nanotoxicity in fish

Christopher Anthony DieniDepartment of Chemistry and Biochemistry

Mount Allison University

UNB Biology Seminar SeriesFriday, March 28th, 2014

Wikimedia Commons

Nanotechnology• Origins traced back to the mid-

20th century• Physicist Richard Feynman

delivers his talk “There’s Plenty of Room at the Bottom” – American Physical Society meeting at Caltech, December 29, 1959

• In more than a half-century since then, we have become dependent on nanotechnology for:• Biosensors• Antimicrobial agents• Drug delivery• Molecular scale electronics• Nanorobotics• … and much more!

Today’s talk

Design of nanomaterials and constituent materials

Synthesis/engineering of nanomaterials

Functionalization/conjugation of nanomaterials for specific purposes (e.g. drug delivery)

Today’s talk

Design of nanomaterials and constituent materials

Synthesis/engineering of nanomaterials

Functionalization/conjugation of nanomaterials for specific purposes (e.g. drug delivery)

Release of nanomaterials in the environment and interaction with indigenous organisms

Nanoparticle toxicity

Nel, A. et al. (2006) Toxic potential of materials at the nanolevel. Science 311: 622-627

Nanoparticle-protein interactions

University of Massachusetts

Model nanoparticle: nanoscale zinc oxide (nZnO)

WebElements.com Wikimedia commons

25 nm nZnO

Dieni et al. Comp Biochem Physiol Toxicol Pharmacol

in press

Scale bar = 1 µm

25 nm nZnO

Dieni et al. Comp Biochem Physiol Toxicol Pharmacol

in press

Scale bar = 1 µm

Wikimedia commons

Uses of nZnO

UK Daily Mail

In vivo exposure In vitro “exposure”

Live physiological

/systemic (e.g.

cardiorespiratory physiology)

Postmortem biochemical

assays(e.g. antioxidant

enzymes, damage markers)

Complex media

(e.g. pooled rat blood plasma)

Simplified conditions

(e.g. BSA solution)

Nanotoxin?

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

The white sucker, Catostomus commersonii

• Benthic (bottom-feeding)

• Likely to come into contact with well-dispersed or sedimentary nanoparticles

• Easily accessible (Silver Lake)

1 mg/L nZnO30 hours

Dr. Tyson J. MacCormackKathryn M. A. Butler, B.Sc.

Biochem (Hons) 2013

Live physiological/systemic level

• Electrocardiography• Respirometry (resting MO2)

• Heart rate decreases by 25% (temporarily)

• No change in resting MO2

Two schools of thought:

• Physiological changes overt enough to affect a whole, live organism are “most meaningful”

• Is a toxic or pathologic response “grave enough?”

• Is a therapeutic “good enough?”

Two schools of thought:

• Physiological changes overt enough to affect a whole, live organism are “most meaningful”

• Is a toxic or pathologic response “grave enough?”

• Is a therapeutic “good enough?”

• Changes at the biochemical level may

not reveal themselves at the systemic level… yet

• Incubation period of an infectious disease before

virulence and immune response

• Initial mutations leading to cancer

• Etc…

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

Reactive oxygen species (ROS)

Superoxide radical anion

Hydroxyl radical

Neal I. Callaghan, Honours Biochemistry

student

OR… others…

Reduced glutathione (GSH)

Oxidized glutathione

(GSSG)

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Glutathione reductase (GR)

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

G6P6PGL Glucose-6-phosphate dehydrogenase (G6PDH)

student

Wikimedia commons

Armstrong JS et al (2004) Bioessays 26: 894-900Wikimedia commons

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Dieni et al. Comp Biochem Physiol Toxicol Pharmacol in press

Control nZnO

G6PDH activity decreased with nZnO

exposure (~29%)

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

GR remained unchanged

Control nZnO

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Control nZnO

Total glutathione levels increased with

nZnO exposure (~56%)

student

Wikimedia commons

student

Wikimedia commons

Control nZnO

c Aconitase activity decreased with nZnO

exposure (~65%)

Reactivated by supplementation with

Fe(NH4)2SO4 (source of Fe2+)

student

Wikimedia commons

student

Wikimedia commons

Armstrong JS et al (2004) Bioessays 26: 894-900Wikimedia commonsDieni et al. Comp Biochem Physiol Toxicol Pharmacol in press

Control nZnO

Malondialdehyde (MDA) levels remained unchanged

RND systems

G6PDH ~29% decreaseGR -

Glutathione ~56% increaseAconitase ~65% decrease

(reactivated by Fe2+)MDA -

Explanation please…?

Hepatic responses to 1 mg/L nZnO exposure

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

G6P6PGL Glucose-6-phosphate dehydrogenase (G6PDH)X

Hydroxyl radical

student

OR… others…

(GSSG)

NADPH NADP+

G6P6PGL Glucose-6-phosphate dehydrogenase (G6PDH)X

XNo activity change,

but deficient NADPH

Increased de novo biosynthesis bringing total levels up

student

Wikimedia commons

student

Wikimedia commons

Aconitase activity decreased with nZnO

exposure (~65%)

MDA levels remained unchanged (?)

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

Patrick T. Gormley, Honours Chemistry

student

Pooled Sprague Dawley rat plasma

Innovative Research

Saline

1% H2O2

1 mg/L nZnO

48 h at 37C

student

Innovative Research

Saline

1% H2O2

1 mg/L nZnO

48 h at 37C

Saline H2O2 nZnO

Ferric reducing ability of plasma (FRAP)

A measure of multiple spontaneously electron-donating antioxidants

Unchanged

student

Innovative Research

Saline

1% H2O2

1 mg/L nZnO

48 h at 37C

Saline H2O2 nZnO

MDA levels remained unchanged

RND systems

student

Innovative Research

Saline

1% H2O2

1 mg/L nZnO

48 h at 37C

Saline H2O2 nZnO

Protein carbonyl levels remained unchanged

Hawkins, CL and Davies, MJ (1998) Biochem J 332: 617-625

1 mg/L nZnO

Decreased hepatic G6PDH activityDecreased hepatic aconitase

activityIncreased hepatic glutathione levels

No plasma changes

In vitro exposureIn vivo exposure

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

No indications of oxidative

damage

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

heart rate

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

G6PDH aconitase

glutathione

Live physiological

/systemic (e.g.

Complex media

(e.g. BSA solution)

Nanotoxin?

G6PDH aconitase

glutathione heart rate

No indications of oxidative

damage

Thank you!Dieni Research Group

Neal I. CallaghanPatrick T. Gormley

MacCormack Lab

Dr. Tyson J. MacCormackKathryn M. A. Butler

Wayne Anderson – Harold Crabtree AqualabJames Ehrman – Mount Allison University Digital Microscopy Facility

Dr. Terry Belke and Jackie Jacob-Vogels – Belke Lab rat blood plasma (initial plasma trials)

Maria Thistle – biostatistics (revisions of latest manuscript)

Marjorie Young Bell FundGoodridge Summer Research Scholarship

Universitas Summer Undergraduate Award

Questions?

cdieni@mta.ca http://chrisdieni.com

http://www.facebook.com/DieniResearchGroup

Dieni CA, Callaghan NI, Gormley PT, Butler KMA, MacCormack TJ. Physiological hepatic response to zinc oxide nanoparticle exposure in the white sucker, Catostomus commersonii. Comp Biochem Physiol Toxicol Pharmacol in press Dieni CA, Stone CJL, Armstrong ML, Callaghan NI, MacCormack TJ. 2013. Spherical gold nanoparticles impede the function of bovine serum albumin in vitro: a new consideration for studies in nanotoxicology. J Nanomater Mol Nanotechnol 2:6

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