navigating environmental public health behind health

a tour of new features

Navigating Environmental Public

Health Behind Health Impacts of Metal

Nanoparticles

Jong Sung Kim, MSc, PhD, Professor & Director of HERC Laboratory

Department of Community Health & Epidemiology

Department of Microbiology & Immunology

Faculty of Medicine, Dalhousie University

HERC Laboratory was established as one of three national centres (UT,

Dalhousie, UBC) of the Canadian Aerosol Research Network (CARN) to

enable a collective study of climate, air quality, population exposures, and

human health. It was created using research awards and matching resources

($4.2 million) from the Canada Foundation for Innovation (CFI), the Nova

Scotia Research and Innovation Trust (NSRIT), Dalhousie Faculty of

Medicine, and industrial partners.

Analytical instrumentation: LC-MS, ICP-MS, GC-MS, HPLC, IC

DALHOUSIE HERC LABORATORY

RESEARCH PROGRAM

To better understand how emerging hazards and exposures lead to

adverse health outcomes at various levels of biological organization

(from cellular and molecular levels to populations) and how human

body modify these responses to maintain homeostasis (host-defense).

Healthy environments for healthy people

WHAT WE DO

• Advance knowledge of the relationship between health and the environment

(evaluating, tracking, and preventing environmental health hazards).

Hazard

Exposure

Health outcome

Dissemination Prevention

Data Stakeholders*

Improved health

*Stakeholders include: Governmental agencies

academia, health care system, policy makers, media

public, business & industry, non-governmental organizations

We have features for every step of the way

WHERE WE LIVE? EMERGING RISKS…

High techrain

High techwashroom

WHERE WE LIVE? EMERGING RISKS…

Nanotechnology is seen as the way

of the future will bring a lot of

benefits nothing is ever perfect!

Cartoon source: www.cartoonstock.com

NANO RISK?Nanotoxicology

WHAT IS NANO?

Source: National Nanotechnology Initiative (NNI)

• “Nano” is a prefix that comes from the Greek word for dwarf.

• It simply means one billionth (1 nm = 10-9 m).

Materials designed and produced to have structural features with

Nanoscale

Think really, really small

1-100 nm

WHAT’S SO SPECIAL ABOUT THE NANOSCALE?

Nanoscale-associated behavior

Scale at which Quantum Effects dominate properties of materials

- The materials’ properties change at the nanoscale (quantum effects).

- Size-dependent properties are the major reason that nanoscale objects have such amazing potential.



Nanoscale-associated behavior

Scale at which surfaces & interfaces play a large role

- Nanomaterials have far larger surface areas than larger-scale materials.

- As surface area of a material increases, a greater amount of the material can come into contact with surrounding materials, thus affecting reactivity.

High Surface area to volume ratio

Surface area increasing

S.A.=h*w*#

• 6 cm2 = (1 cm)2 * 6

• 60 cm2 = (1/10 cm)2 * 6 * 1000

• 6*107 cm2 = (1/10 cm)2 * 6 * 109



Higher Surface area to volume ratio

NMs can be made to be stronger, lighter, more durable, water-repellent, antimicrobial, self-cleaning, better electrical conductors among other traits.

Benefits of Small Systems

Doing more with less

Multi-functionality

New applications

Quicker performance

Better performance

NANOTECHNOLOGY APPLICATIONS


NT Applications

US $76 billion by 2020

POTENTIAL HEALTH IMPACTS

The Bio-Nano Interface: Nanoparticle-Bio Interactions

Scale at Which Much of Biology Occurs

- Hemoglobin, the protein that carries oxygen through the body,

is 5.5 nm in diameter.

- A strand of DNA, one of the building blocks of human life, is

only about 2.5 nm in diameter.

Interfacing engineered NPs with biological systems:

Anticipating adverse nano-bio interactions


POTENTIAL HEALTH IMPACTS

Risk = Function of Hazard and Exposure.

Human exposure to NPs: skin (dermal), lungs (inhalation), gastrointestinal

tract (ingestion), or by injecting as a formulated medicine.

The most critical concern over health & environmental effects: when NPs

are aerosolized (highly mobile & enter the human body via inhalation).

Source: Card et al (2008) Am J Physiol Lung Cell Mol Physiol 295: L400-411.

THE CHALLENGE OF TESTING NP TOXICITY

Toxicity test methods, which are routinely applied to testing of nanomaterials, were originally developed for soluble chemicals.

Nanomaterials are different than chemicals.

We should not treat them as chemicals.

Nanomaterials are fundamentally different from many 'conventional' chemicals as they often have limited or no solubility at all and are potentially released to the environment.

Only limited nano-specific guidance on ecotoxicity testing is currently available (OECD)

TOXICITY TESTING MODELS

In Vitro (Cells) Study

In Vivo (Animal) Study

Nanomaterials

Nanoscale-

associated behavior

Human

DESIGN CRITERIA

Design Criteria:

Toxicity Testing of Environmental Agents

Toxicity Testing in the 21st Century: a vision and a strategy. US National Research Council of the National Academies 2007

OPTIONS FOR FUTURE TOXICITY TESTING STRATEGIES

Toxicity Testing in the 21st Century: a vision and a strategy. US National Research Council of the National Academies 2007

PULMONARY TOXICITY ASSESSMENT

In Vivo Model (using animals)

Allow realistic route with reproducible NP dosing & lung

distribution

Inhalation & instillation exposures

Bronchoalveolar lavage fluid evaluation

• Cell differential analysis

• Lactate dehydrogenase

• Cytokine/chemokines

General Components of the Pulmonary Bioassay

Lung tissue analysis

• Lung histopathology

• Dosimetry

• Enzyme activity

BRONCHO ALVEOLAR LAVAGE (BAL)

Cytokines/chemokines, total protein &

lactate dehydrogenase (LDH)

Saline solution

Fluids Cells

Total cell & Differential cell counts

(macrophages, neutrophils, lymphocytes etc)

A saline wash of the

airways (broncho) and air

sacs (alveolar) for

recovery of inflammatory

cells.

Indicate inflammatory

responses and

cytotoxicity induced by

toxicant

IN VIVO NANOTOXICOLOGY

• Human exposure to NPs & environmental bacteria can occur

simultaneously.

• The purpose of this study was to determine if host defense

against bacterial infection is enhanced or impaired by Cu NPs in

a murine pulmonary infection model.


Schematic of the pulmonary bacterial

clearance model. We established a murine

pulmonary infection model of Klebsiella

pneumoniae (K.p.) to determine if pulmonary

bacterial clearance is impaired by NP

exposure. Following both sub-acute inhalation

and intratracheal instillation, mice were

intratracheally challenged with K.p. bacteria at

a dose of 1.4 ± 0.1 × 105 CFUs/mouse.

DOSIMETRY (ICP-MS)

IN VITRO MODEL IN NANOTOXICOLOGY

In vitro assays can serve as a screening method for assessing NP

toxicity.

opportunity for extensive investigation of

(can’t be conducted in vivo).

Alternative and the 3 Rs (Replace, Reduce, Refine)

Submerged System

Particle suspension in medium & Exposure of

immersed cells

In Vitro Exposure of Lung Cells to Nanoparticles

Air-Liquid Interface (ALI)

Air delivery of NPs to lung cells at the ALI

NP INTERACTION WITH ALVELOAR EPITHELIUM

Source: Card et al (2008) Am J Physiol Lung Cell Mol Physiol 295: L400-411.

into the systemic circulation is

most likely to across with its very large

surface area (>100 m2 in humans) and thin barrier thickness.

However, interactions between

LIMITATIONS: IN VITRO STUDY

Medium

Proteins

Cells

Agglomeration

Air-Liquid Interface Exposure

NPs

Basolateral side: Medium

Airway Surface Liquid (ASL)

Apical side: Lung cells

Semi-permeableMembrane

Exposure chamber

Transwell

Drawbacks • Not mimicking alveolar epithelial conditions in vivo.

• Interaction between NPs & media.

• Uncertainty of dosimetry.

• NP agglomeration/dispersion problems.

Conventional in vitro study: Need new approach

IN VITRO NANOTOXICOLOGY

• The objective of this study was to overcome the limitations of

conventional in vitro exposure of submerged lung cells to NPs for

NP toxicity assessment.

• We developed a dynamic in vitro exposure system (DIVES) capable

of generating and depositing airborne NPs directly onto lung cells at

an ALI (simulation of human pulmonary exposure to NPs).

Aerosol Inlet

Cell culture insert

Cells on membrane

Culture medium

Air o

ut

Air o

ut

Air

Cellular Responses

NANOPARTICLE IN VITRO EXPOSURE SYSTEM

Figure: A Schematic of a NP In Vitro Exposure System. This in vitro approach simulates particle deposition in the human lung more realistically than does submerged cell exposure(without an apical air interface), and it preserves the inherent properties of the particles.

(Vitrocell)

NANOPARTICLE IN VITRO EXPOSURE SYSTEM

NANOPARTICLE SIZE DISTRIBUTION

CELL VIABILITY (AB ASSAY)

INTRACELLULAR ROS

CELL VIABILITY (Fe vs Cu NPs)

INTRACELLULAR ROS (Fe vs Cu NPs)

CELLULAR DOSIMETRY (ICP-MS)

Table: The mass concentration of Cu after exposure of A549 cells to Cu NPs at the ALI.

Adjusted Cu massin/on the cells

Adjusted Cu massin basal medium

Total air-delivered Cu mass concentration

(4.7 cm2) (18 mL) (Transwell) (µg NP/cm2)

---------- µg ± SE ----------

1.7 ± 0.1 2.9 ± 0.1 4.6 ± 0.1 1.0 ± 0.02

Cellular Dosimetry: large amount of Cu was dissolved and released to the

medium (62% of total mass) during continuous air-delivery of Cu NPs.

Digest A549 cells + Cu NPs

HCl + HNO3 at 95 °C (~ 4 h)• Measure deposited Cu by ICP-MS

• [Cells + Cu NPs] – [Cells]

• A549 cells (a human alveolar type-II-like cell line)• Type II alveolar epithelial cells: first lung cells to be exposed to inhaled NPs.

CELLULAR DOSIMETRY (ICP-MS)

NP TOXICITY RANKING (IN VITRO VS. IN VIVO)

S.P DISEASE BURDEN

Most people have colonization of S.p in nasopharynx, sinuses, nasal cavities, and have no

symptoms.

Infants, elderly, and chronically ill at highest risk for pneumonia, and other diseases.

Pneumonia (and related diseases) result in more than 4 million deaths per year (mostly by

S.p).

Big picture question: What are the underlying mechanisms that make S.p pathogenic

and cause disease? What makes certain people/groups susceptible?

Member of the streptococcus family

Gram positive diplococci

NPs in Welding Fume

†Graczyk, H., et al., Characterization of Tungsten Inert Gas (TIG) Welding Fume Generated by Apprentice Welders. Annals of

Occupational Hygiene, 2016. 60(2): p. 205-219.

A recent welding fume characterization study at the

breathing zone across welders (n=20)† indicated 92%

of the particles were <100 nm, with 50% of the

particles <41 nm. Inhalation of these metal fume

NPs can result in particle deposition onto the alveolar

epithelial surface of the lungs, compromising the

respiratory and circulatory systems

Exposure to welding fumes increase the risk of S.p infections in welders.

Pneumonia was associated with a reported occupational exposure to metal fumes in

the previous year (OR = 1.96).

To date, the mechanism by which welding fumes increase susceptibility to S.p

infection is not well known.

Are the metal NPs in the welding fumes to blame?

S.P DISEASE BURDEN

Goal: to determine the relationship between metal NP exposure (e.g.: copper) and the

increased susceptibility of welders to infection by Streptococcus pneumoniae.

Do copper NPs promote cytotoxicity in human alveolar epithelial cells (A549 cells) to

enhance adhesion of S.p?

S.p adhesion to lung cells is increased after Cu NP exposure

FUNDING SOURCES

The Power of Innovation!


Contact:Dr. Jong Sung Kim, Laboratory DirectorEmail: [email protected]: 902-494-4225

mailto:[email protected]

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