RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

The literature pertaining to the present study on “Pulmonary Function and

Markers of Oxidative Stress and Inflammation in Photocopier Operators” is reviewed

under the following heads:

2.1 Indoor Air Pollution

2.2 Photocopiers and their Emissions

2.3 Health Effects of Exposure to Photocopier Emissions

2.4 Indoor Air Pollutants and Pulmonary Function

2.5 Biomarkers of Pulmonary Damage

2.6 Metabolomics as a Tool to Assess Pulmonary Function

2.1 Indoor Air Pollution

Pollution has been defined as the direct or indirect introduction of substances into

the environment that harms living resources, affects human health and impairs

environmental quality. Atmospheric air pollution is defined as any gaseous or particulate

matter in the air that is not a normal air constituent or is not normally present in the air in

high concentrations (Yang and Omaye, 2009).

Kampa and Castanas (2008) defined air pollutant as any substance which may

harm humans, animals, vegetation or material. Though a number of physical activities

(volcanoes, fire, etc.) release different air pollutants into the environment, anthropogenic

activities are the major causes of environmental air pollution. Hazardous chemicals can

escape into the environment by accident as well as from industrial facilities and other

activities.

According to Ko and Hui (2012), exposure to some degree of outdoor air

pollution is unavoidable during the entire life span. However, Hoskins (2011) argues that

the indoor environment might be greatly polluted than the outdoor environment. In

2

accordance with the above, the World Health Organization (WHO, 2012) estimated that

outdoor air pollution might contribute to 1.3 million deaths per year world-wide (5% of

deaths) whereas indoor air pollution might lead to 2 million premature deaths in

developing countries.

Colbeck et al. (2010) opined that population exposure to various air pollutants is

likely to be higher in the indoor micro-environment than outdoors due to the amount of

time people spend there. People spend approximately 80% of their time in indoor

environment (Bai et al., 2010). Hence, Singh and Jamal (2012) considered indoor air

pollution to be five times more hazardous than outdoor air pollution.

Among the indoor environments we live in, work place plays an important role.

The number of people working in indoor environments for their livelihoods is increasing

day by day. Hence, occupational exposure to air pollutants in indoor environments is of

prime concern. However, extensive research has not been carried out on occupational

exposure to air pollutants in non industrial settings.

With the rapid development of information technology, the affiliated output

equipment, which is mainly composed of laser printers, inkjet printers, multi-functional

photocopiers and so on, has become the third largest information technology market.

Printers and copiers have become standard indoor electronic equipment (Saraga et al.,

2011). The worldwide demand for copiers in 2010 was approximately 1 million units.

Narita and Obinata (2011) envisaged that the market for electrophotography based

printers and copiers might grow at an annual rate of approximately 8%.

According to Yang and Haung (2008), thousands of people across the world are

involved in the operation of photocopiers either in commercial photocopier units or

offices. Working with photocopiers has been shown to be associated with an increased

prevalence of sick building syndrome symptoms for some time. Consequently, the

effects of photocopier emissions on human health have received considerable attention

in recent days.

From the great deal of research that has been conducted into the response to air

pollutant exposure at the molecular, cellular and whole organism level, Holloway et al.

(2012) surmised that there may be significant differences in biological responses

induced by differing types of air pollutants derived from a variety of sources.

2.2 Photocopiers and Their Emissions

Photocopying is the process of producing copies of original documents and

drawings by exposing the originals to chemicals, light, heat or electrostatic energy and

recording the resulting images on a sensitized surface. Xerography was developed by

the American physicist Chester F. Carlson in 1938. In 1947, the Haloid Company (later

renamed Xerox) of Rochester, New York, USA, obtained the commercial rights to

Carlson's invention. Thirteen years later, the company, introduced its first office copier.

Many other companies now manufacture similar machines (Bhattacharjee and Kar,

2004).

2.2.1 Photocopying Process

Photocopying was originally called as electrophotography. This technology is

used to produce high-quality text and graphic images on paper. The six-step process in

photocopying is as follows:

1. A photoconductive surface is given a positive electrical charge.

2. The charged photoconductive surface is exposed to the image of a document.

The non-image areas become more conductive, the charge dissipates.

The image areas are positively charged; negatively charged powder spreads

over the surface adheres through electrostatic attraction.

3. A piece of paper is placed over the powder image and then given a positive

charge.

4. The powder is attracted to the paper when it is separated from the

photoconductor.

5. Heat fuses the powder image to the paper.

Review of Literature

6. The photoconductor is cleaned to restart the process (Hays, 2003).

Figure 1

The working principle of a photocopier

Evans et al. (2006a)

Palanna (2010) acknowledged that nowadays digital photocopiers are

extensively used. They are equipped with light sensitive electronic cells called charge

coupled devices. When copying starts, the cells respond to the light reflected from the

original and convert it into a digital code of electronic signals. These signals can be

used to print a copy directly, guiding a laser beam to build up the image line by line. The

images can also be stored in the photocopier’s memory. Precise processes may differ

depending on the machine being used. Modern photocopiers can make up to 135

copies every minute and copy hundreds of pages with just one touch of a button. They

can also make images larger, smaller, darker or lighter than the original. Many can print

copies on both sides of a paper, feed in originals automatically, sort copy sheets into

separate piles and staple the copies together (Figure 1).

2.2.2 Photocopier Toners

Review of Literature

Figure 2

Electron microscopic image of

toner particle

Duke et al. (2002)

Toner = 10 m toner; Carrier = 100 m

Toner is one of the largest consumables in daily office work. Its demand is

increasing with the popularity of printers and photocopiers. It is estimated that the global

demand for toner is around 2, 40, 000 - 2, 60, 000 tons (Bai et al., 2010).

Oberdorster et al. (2005) and Abraham et al. (2010) stated that toners are made

by a mechanical process involving melt mixing and grinding of the raw materials to

produce a fine powder; followed by the addition of surface additives. Mean particle sizes

resulting from this process are in the range of 5-10 µm. In 2002, photocopier

manufacturer Xerox introduced a new chemical process to grow toner particles,

resulting in smaller particles of a more

uniform shape, smoother surface and

narrower size range.

Kojima and Elliott (2012) stated that toners

are composed of resin particles (usually

styrene - acrylate) functioning as colouring

material and carrier particles, usually made of

magnetic materials such as iron oxide or

carbon black with a mean diameter of 50 – 100

μm (as illustrated in Figure 2). The function of the

carrier magnetic particles is to add

electrostatic charge to the toners and carry the

toners onto the latent images. According to Adetunji et al. (2009) charge control agent,

flow control additives such as fumed silica, titanium oxide, organometallic salts and wax

are also found in toners.

Gminski et al. (2011) observed that colour toners contained various organic

pigments. They also suggested that impurities, namely Polycyclic Aromatic

Hydrocarbons (PAHs) and heavy metals may be present in carbon black and iron oxide.

Table 1 presents the components present in photocopier toners.

Table 1

Components present in photocopier toners

Review of Literature

Components present in toners Reported by

Magnetite, Silicon, Iron, Nickel, Zinc, Arsenic, Cadmium,

Antimony, Lead, PAH

Gminski et al. (2011)

Iron oxide, amorphous silica, paraffin wax, polymethyl

methacrylate, diethylene glycol, 2-pyrrolidone

Nelson et al. (2011)

Iron, Titanium, Silicon, Manganese, Sulphur, Tin,

Aluminium, Zinc, Magnesium, Copper, Chromium,

Molybdenum, Nickel and long chain alkanes

Bello et al. (2012)

Iron, Aluminium, Silicon Tang et al. (2011)

Titanium, Vanadium, Iron, Manganese, Chromium, Zinc,

Cobalt, Nickel, Arsenic, Yttrium

Bai et al. (2010)

Silicon, Sulphur, Titanium, Iron, Chromium, Nickel, Zinc,

Chlorine, Calcium

Barthel et al. (2011)

2.2.3 Photocopiers are Sources of Indoor Air Pollutants

Bar-Sela and Shoenfeld (2008), Balasubramanian (2010) and Pirela et al.

(2012) have confirmed the earlier reports that photocopiers are potential sources of

indoor air pollution. Photocopiers have been recorded to emit ozone, nitrogen oxides,

volatile organic compounds (VOCs), semi volatile organic compounds (SVOCs), air

borne particles including nanoparticles, small doses of UV light, metals like selenium,

cadmium and arsenic and extremely low frequency electromagnetic fields.

Consequently, the effects of photocopiers on indoor air quality have received

considerable attention nowdays.

He et al. (2007 and 2010), Schripp et al. (2008) and Morawska et al. (2009)

opined that photocopier emissions were highly variable and depended on a host of

poorly understood factors such as photocopier’s manufacturer, model, printer age, fuser

temperature, page coverage and printing frequency. According to Lee and Hsu (2007) a

range of other materials in photocopy centres, such as printed documents, cleaning

solvent, office furniture, building materials, flooring materials and other office equipment,

might also emit chemicals into the indoor environment.

In addition, photocopiers might be housed in small rooms without sufficient

ventilation. Maddalena et al. (2011) felt that emission rates of air pollutants from office

equipment might be relatively low, but they might be potentially important sources of

exposure due to their very close proximity to people. Kleinsorge et al. (2011) suggested

that proximity might be particularly important in the case of SVOCs, ultrafine particles

(UFPs) and reactive species that could interact with different surfaces where rapid

transformations occur. The mode of operation of photocopiers could influence the

temperature and air circulation under ambient conditions, which in turn could impact the

emission rates of pollutants.

2.2.3.1 Particulate Matter

Emmerechts and Hoylaerts (2012) defined particulate matter (PM) as a

heterogeneous mixture of solid and liquid particles suspended in air. They categorized

PM based on size range into thoracic particles (PM10) with a mean aerodynamic

diameter <10 μm, coarse particles >2.5 μm and <10 μm, fine particles (PM2.5) <2.5 μm

and ultrafine particles (UFP) <0.1 μm.

Bello et al. (2012) stated that the term ‘ultrafine particles’, is synonymous with

incidental nanoparticles. The term ‘nanoparticle’ refers to particles with three

dimensions in the nanoscale (1–100 nm) range. When nanoparticles are generated as

unintended by-products of industrial or anthropogenic activities they are generally called

incidental nanoparticles (eg. diesel exhaust or welding fumes). By contrast, engineered

nanoparticles are intentionally produced to fulfil desired technological functions (eg.

carbon black or titanium dioxide). Photocopier toners contain engineered nanoparticles

whereas emissions from photocopier operations might fall into the category of incidental

nanoparticles.

According to Destaillats et al. (2008), significant levels of particulate matter are

generally found during operation of printers, copiers and multi-functional devices.

McGarry et al. (2011) reported that, of the particles emitted, fine particles and

particularly ultra fine particles are predominant. Numerous other studies in different

parts of the world have found similar results (Kagi et al., 2007; Lee and Hsu

2007; Wensing et al., 2008; Adetunji et al., 2009; Morawska et al., 2009; Schripp et al.,

2009 and He et al., 2010).

Tang et al. (2011) proposed that particle concentrations might be affected by

various factors, namely, printer, toner, paper type, maintenance cycles and air

exchange rate. Similar conclusions have also been drawn by Wensing et al. (2008) and

Schripp et al. (2009).

In addition Schripp and Wensing (2009), Morawska et al. (2009) and Maddalena

et al. (2011) concurred that the heated toner powder, ink, paper, fuser roller and

lubricant oil were all potential sub micrometer particle sources. Experiments by Wensing

et al. (2008) and He et al. (2010) showed that fuser roller temperature was the principal

factor governing the rate of particle formation in laser printer operation.

According to Bello et al. (2012), chemical composition of all airborne PM fractions

emitted by photocopiers was reported to be complex. They contained all major elements

and classes of analytes found in the toners, including metals (fumed silica, titanium

dioxide and iron oxide), SVOCs, organic and elemental carbon, implicating different

sources.

Barthel et al. (2011) reported the emission of other elements, including silicon,

sulphur and several metals such as titanium, iron, chromium, nickel and zinc. They

estimated that solid inorganic particles accounted for <2% of the total number of emitted

particles.

Fan et al. (2003 and 2005) and Lee and Hsu (2007) opined that secondary

organic aerosol (SOA) formation inside photocopiers might be an important source of

indoor UFPs during photocopying. SOA and UFPs would have been formed when

ozone reacts with unsaturated VOCs (such as terpenes and styrene). Ions generated by

corona devices during photocopying, might play a role in the formation of UFP by ion-

induced nucleation of organic vapours.

2.2.3.2 Ozone Emissions

Ozone (O3) is a highly reactive, colourless-to-bluish gas with a characteristic

odour associated with electrical discharges. Brook et al. (2004) connoted that low-level

exposure to ozone is ubiquitous, because O3 is formed by natural processes and by

human activities.

Photocopiers have been recorded to emit ozone as early as 1978 (Allen

et al., 1978). Several reports over the last decade have ensured ozone emissions by

photocopiers (Tuomi et al., 2000; Stefaniak et al., 2000; Kagi et al., 2007;

Bo and Chen, 2010; Tipayarom and Tipayarom, 2011 and Wang et al., 2012).

Valuntaite and Girgzdiene (2007) suggested that ozone is generated from the

reaction of charged ions and electrons with atmospheric gases during the

electrophotographic process of the copier and from the corona wire (coil that serves a

positive charge in the surface of the drum of the copier). It has been established that

ozone concentration increases with the intensity of the copying process and decreases

with the distance from a copying machine. Tipayarom and Tipayarom (2011) opined that

ozone might also form from ultraviolet radiation sources in copier units. Balakrishnan

and Das (2010) cautioned that ozone could reach dangerous levels in small and poorly

ventilated copying rooms.

Destaillats et al. (2008) observed that even low levels of ozone can react with

other indoor pollutants to produce secondary pollutants of ultra fine aerosol particles.

However, Ewers and Nowak (2006) stated that, in contrast to older devices

where the imaging drum is electrostatically charged with a corona wire, modern laser

printers and copiers emit considerably smaller amounts of ozone as most of them

usually employ the transfer-roller-technology.

2.2.3.3 Volatile Organic Compound Emissions

VOCs are organic substances which are volatile and are photo-chemically

reactive. VOCs are part of the large hydrocarbon family consisting of a vast array of

aliphatic, aromatic hydrocarbons, their halogenated derivatives, alcohols, ketones and

aldehydes. They are highly reactive and participate in atmospheric photochemical

reactions. In the presence of oxides of nitrogen and sunlight, VOCs form ozone and

other products. Oxidation of VOCs by reaction with hydroxyl radicals is the main

removal process. The oxidation of complex organic molecules might lead to the

fragmentation and production of a range of reactive free radicals and more stable

smaller molecules such as aldehydes (Srivastava and Mazumdar, 2011).

The emission of VOCs by photocopy machines and laser printers has been

clearly documented by Lee et al. (2006), Uhde et al. (2006), Wensing et al.

(2006), Kagi et al. (2007) and Lee and Hsu (2007). In concurrence with their reports,

Henschel et al. (2003), Lee et al. (2006), Hsu et al. (2005 and 2006), Destaillats et al.

(2008), Balakrishnan and Das (2010) and Maddalena et al. (2011) confirmed

the presence of benzene, toluene, ethyl benzene, isopropyl benzene, xylenes, styrene,

acetophenone, methyl styrene, 1, 2, 4 trimethyl benzene, butyl benzene, methoxyethyl

benzene, alkanes such as isodecane, 2, 2, 4 trimethyl octane, aldehydes such as

formaldehyde, acetaldehyde and benzaldehyde, nitropyrene, phthalates and

isocyanates, as well as, an array of other substituted benzenes along with a number of

flame retardants such as the polybrominated diphenyl ethers (brominated flame

retardants) and organophosphorus compounds. A conceptual model of indoor air

chemistry and particle formation and removal during the process of photocopying is

presented in Figure 3.

Figure 3

Conceptual model of indoor air chemistry and particle formation and removal during photocopying

Review of Literature

Lee and Hsu, 2007 Depending on the characteristics of the toner and the fuser materials, the toners

consistently emit VOCs. Photocopiers are designed to maintain at a relatively high

temperature in idle mode, in order for immediate operation. Thus, VOCs will be emitted

whenever photocopiers are powered, regardless of whether photocopiers are in

operation or not. VOCs may be emitted from paper that is heated in the fuser of a dry-

process copier (Lee et al., 2006; Hsu et al., 2005, 2006 and Lee and Hsu, 2007). As

already stated, ozone reacts with printer-generated VOCs to form secondary organic

aerosols (SOAs) (Wang et al., 2012).

According to Destaillats et al. (2008) emissions of VOCs from printers and

copiers are generally higher than from computers, particularly styrene, toluene, xylene

and other alkyl benzenes. Lee et al. (2006) found that the concentrations of benzene,

toluene, ethyl benzene, xylenes (BTEXS) and styrene were well below the occupational

exposure threshold guidelines. However, under conditions of inadequate ventilation, it is

feasible that individuals working in the photocopy centers may be exposed to high levels

of VOC.

2.2.3.4 Semi Volatile Organic Compounds

Semi volatile organic compounds (SVOCs) are defined as compounds with

boiling points ranging from 240° – 260° C to 380° – 400° C with polar compounds in the

high end of the intervals. SVOCs may be distributed between the gas phase and the

particle phase in indoor air (Clausen and Kofoed-Sorensen, 2009). Xu and Zhang

(2011) warned that SVOCs constitute an important class of indoor air pollutants and

include phthalate esters, brominated flame-retardants, polychlorinated biphenyls,

nonionic surfactants and pesticides.

Wensing et al. (2008), Morawska et al. (2009), Maddalena et al. (2011) and Bello

et al. (2012) concluded that printers and photocopiers emitted a range of SVOCs such

as phthalate esters, brominated flame retardants, organophosphate flame retardants,

polycyclic aromatic hydrocarbons and perfluoroalkyl compounds.

Corona devices/Light O3, Nitrogen oxides, ions,

OH radicals & h

2.2.3.5 Toner Emissions Bai et al. (2010) reported that toners are fixed onto the paper by fusion at about

170°C. According to their study, about 75% of the toner is transferred to the

photoconductive drum. Toner particles that do not adhere to the drum might be

available for emission in the indoor air.

Chamber investigations and indoor air measurements by Uhde et al. (2006),

Wensing et al. (2006 and 2008), He et al. (2007), Kagi et al. (2007), Schripp et al.

(2008) and Tang et al. (2011 and 2012) have shown that fine particles as well as sub-

micrometer particles of toner and paper are released into indoor air during printing.

In contrast, Lee and Hsu (2007) found that the emitted particles were much

smaller than the original toner powders. Tang et al. (2011) proposed that fragments or

aggregates melted in the fuser unit were released into the indoor air rather than whole,

undamaged toner particles. This is in accordance with the results obtained by

Morawska et al. (2009), Caesar and Schmitt (2009), Barthel et al. (2011) and Bello et al.

(2012).

He et al. (2007) investigated the particle emission characteristics of office printers

and found that the particle emission rates were printer-type specific and were affected

by toner coverage and cartridge age.

2.2.3.6 Selenium Emissions

The photoreceptor drum or belt is an important part of the photocopier. It is

basically a metal roller covered by a layer of photoconductive material. This layer is

made up of a semiconductor such as selenium, germanium or silicon (O’Connell, 2001).

Bar-Sela and Shoenfeld (2008) and Palanna (2010) observed that small amounts of

selenium, arsenic or cadmium oxide might also be found in the room.

George and Wagner (2009) and Boyd (2011) reported that prior to the 1990s, the

photocopier manufacturer, Xerox used arsenic, selenium and tellurium in photocopiers.

In the early 1980s, changes in photoreceptor design enabled Xerox to replace arsenic

and selenium with new organic materials. Hence, selenium is rarely used in

photocopiers these days.

2.3 Health Effects of Exposure to Photocopier Emissions

Air pollution has been associated with numerous adverse health outcomes. It

causes acute effects such as respiratory symptoms, cardiovascular events, hospital

admissions and mortality. Long-term exposures to air pollution have been associated

with chronic bronchitis, markers of atherosclerosis, lung cancer and mortality (Gotschi et

al., 2008).

According to Weschler (2006), in the non-occupational indoor setting,

environmental exposures are often more subtle and not readily recognized. In the most

extreme cases, controversial terms like sick building syndrome, toxic mold syndrome

and multiple chemical sensitivity have been coined for lack of a better way to

characterize unexplained constellation of symptoms that are attributed to exposure in

the home or nonindustrial occupational settings.

Destaillats et al. (2008) opined that personal exposures to pollutants emitted by

photocopiers might be significantly larger than those estimated through average

pollutant indoor concentrations, due to proximity of users to the sources over extended

periods of time.

Yang and Haung (2008) declared that occupational exposure to pollutants

emitted from photocopiers was not significantly associated with an excess of chronic

respiratory symptoms and acute irritative symptoms in photocopy employees. They

were of the view, that the current exposure levels in photocopy centers may be

sufficiently safe in well-controlled work environments, especially if the photocopier is

handled carefully.

2.3.1 Particulate Matter

Coccini et al. (2012) reported that particulate matter (PM) is related to the most

serious effects of air pollution on human health, because particles contain a broad

range of toxic substances. Pope and Dockery (2006) contemplated that PM in ambient

air might be an important risk factor for acute and long-term adverse effects related to

pulmonary and cardiovascular diseases, cancer and mortality. According to

Lambert et al. (2003), PM has also been shown to exacerbate a variety of pulmonary

disorders, including chronic obstructive pulmonary disease, asthma and lower

respiratory tract infections

Li and Nel (2011), Jackson et al. (2012) and Emmerechts and Hoylaerts (2012)

felt that the predominant route of exposure to ultrafine particles in the working

environment is through inhalation. However, Adetunji et al. (2009) disagreed with them,

suggesting that engineered nanoparticles might penetrate the human body through

inhalation as well as ingestion, skin and injection.

Daigle et al. (2003) and Frampton (2007) proposed that large particles

demonstrated a great fractional deposition in the extra thoracic and upper trachea-

bronchial regions, whereas small particles (e. g. PM2.5) showed great deposition in the

deep lungs. By virtue of their small size (<0.1 μm), ultra fine particles (UFPs) might

penetrate deep into the lungs and deposit in the alveoli.

Geiser et al. (2005) argued that UFPs might evade alveolar macrophage

clearance from the lung and enter lung cells, the interstitium and possibly the vascular

bed. Elder et al. (2006) and Samet et al. (2009) were under the impression that UFPs

might travel from the lung through blood and lymphatic circulation to other organs

including bone marrow, lymph nodes, spleen, heart and even the brain through axonal

transport in the olfactory nerve.

Li et al. (2003), Delfino et al. (2005), Oberdorster et al. (2005), Nel et al. (2006),

Xia et al. (2009), Araujo and Nel (2009) and Coccini et al. (2012) concurred that among

the different particle sizes, UFPs might be potentially the most dangerous. They might

have greater potential to induce oxidative stress and inflammation and might be more

pro-atherogenic than larger particles owing to their small size, higher particle number

concentration, deep penetration, higher air way deposition efficiency, high content of

redox cycling organic chemicals, large surface area / volume ratio, transition metal

components and high rates of retention in the lung.

Figure 4

Interaction among air pollutants and effects on cardio vascular health

NOx-

Nitrogen oxides; SVOC – Semi volatile organic compounds; CO – Carbon monoxide; O3 – Ozone; PM – Particulate matter; COPD – Chronic obstructive pulmonary disease; CV – cardiovascular; ↑ BP – increased blood pressure; 2° - secondary; ACS – Acute coronary syndrome

Brook and Brook (2011) Knaapen et al. (2004) suggested that the mechanism of action of PM might

involve inflammation and oxidative stress. Jackson et al. (2012) were of the view that

the biological and pathological consequences of pulmonary exposure to nanoparticles

including oxidative stress, DNA damage, fibrosis, cardiovascular events and lung

diseases were driven primarily by inflammation. In concurrence, Larsson et al. (2007),

Review of Literature

Nystrom et al. (2010), Strak et al. (2010) and Zuurbier et al. (2011) showed that

increased systemic markers of inflammation and signs of lower airway irritation and

decreased lung function were found after exposure to UFPs.

However, according to Xia et al. (2009), oxidative stress might be a key injury

mechanism that relates to pro-inflammatory and pro-atherogenic effects of UFPs

in the respiratory and cardiovascular tracts, respectively. Cardiovascular and respiratory

inflammation resulting from the induction of oxidative stress could play an important role

in disease pathogenesis. They suggested three different possibilities for the

development of cardiovascular and respiratory inflammation from oxidative stress

namely, inhaled particles releasing organic chemicals and transition metals from the

lung to the systemic circulation, pulmonary inflammation leading to the release of

reactive oxygen species (ROS), cytokines and chemokines to the systemic circulation,

UFPs entering the systemic circulation by penetrating the alveolar / capillary barrier in

the lung (Figure 4). Li et al. (2003) and Nel (2005) corroborated the above suggestions

by stating that in response to UFP uptake, target cells such as airway epithelial cells

and macrophages might also produce ROS during biologically catalysed redox

reactions.

Emmerechts and Hoylaerts (2012) proposed that inhaled particles might affect

the cardiovascular system through three pathways: interference with the autonomic

nervous system, direct translocation of UFP into the systemic circulation and pulmonary

inflammation associated oxidative stress.

Hoffmann et al. (2009), Coccini et al. (2012) and Jackson et al. (2012) affirmed

the translocation of particles from the lungs into blood circulation and extra-pulmonary

organs. The ability of UFPs to translocate systemically from pulmonary sites might

make them particularly relevant to the cardiovascular effects of inhaled PM. Particles

deposited in the alveoli might cause airway injury or activation of blood cells (such as

monocytes) and lead to release of pro-inflammatory cytokines interleukin (IL)-6 and IL-

8. Increased production of IL-6 and IL-8 might activate mononuclear as well as

endothelial cells, initiating the hepatic synthesis of acute-phase proteins, such as C

Reactive Protein (CRP) and serum amyloid A. These might upregulate adhesion

molecules expression (e.g. E-selectin, von Willebrand factor antigen, ICAM-1).

Increased expression of adhesion molecules are markers of endothelial dysfunction. An

enhanced acute-phase response as well as endothelial cell activation might increase

procoagulant activity, indicated by a rise in coagulation proteins (e.g. fibrinogen,

factor VII, prothrombin fragment 1+2, D-dimer) and evidence of the clotting cascade

activation. These changes in blood parameters, together with plaque instability might

ultimately lead to a coronary event.

Zhou et al. (2003), Gadhia et al. (2005) and Kleinsorge et al. (2011) observed

elevated oxidative stress levels among workers in photocopier units. Khatri et al. (2012)

found short term exposure to photocopier emissions resulted in higher oxidative stress

and local airway inflammation.

Oxidative DNA damage was positively correlated with cumulative UFP

exposure by Vinzents et al. (2005). Significant DNA damage was observed among

photocopier workers by several investigators (Goud et al., 2004; Gadhia et al.,

2005; Manikantan et al., 2010; Balakrishnan and Das, 2010 and Kleinsorge et al., 2011)

2.3.2 Ozone

Wolkoff et al. (2006) and Maddalena et al. (2011) associated ozone exposure

with occupational symptoms such as eye, nose or throat irritation, headache and

fatigue. Studies by Blankenberg et al. (2003), Arjomandi et al. (2005), Alexis et al.

(2010) and Kim et al. (2011) have proved that O3 exposures initiate and exacerbate

pulmonary disease. They also suggested it might lead to lung inflammation and

significant decrements in lung function. Brown (2009) asserted that the magnitude of

respiratory symptoms, pulmonary function decrements and inflammatory responses

were generally a function of O3 concentration, minute ventilation and exposure duration.

According to Schegele et al. (2009), responsiveness to ozone was quite variable

among people, but reproducible within individuals. With repeated daily exposures, the

declines in lung function were attenuated, but other markers of airway inflammation and

injury persisted or increased. Smokers were reported to experience minimal lung

function effects, but remained susceptible to the airway inflammatory effects.

Epidemiological studies have shown associations between O3 exposure and

increased cardiovascular morbidity and mortality. Pulmonary and cardiovascular

systems are intimately coupled. Therefore, the diverse biochemical alterations caused

by O3 generated bioactive reaction products might activate immune cascades within the

lung and initiate downstream cardiovascular perturbations. Preliminary studies have

reported that ozone exposure might significantly alter blood pressure and vascular

function, heart rate, endothelial-dependent vascular function, oxidative stress,

mitochondrial damage and atherogenesis (Chuang et al. 2009).

Palli et al. (2009) reported that ozone concentrations modulated oxidative DNA

damage in circulating lymphocytes. In tune with the same, elevated DNA damage has

been reported among photocopier workers exposed to ozone by Gadhia et al. (2005),

Goud et al. (2004), Kleinsorge et al. (2011), Manikantan et al. (2010) and

Balakrishnan and Das, (2010).

Ozone is a highly reactive gas. Consequently, ozone might react with other

pollutants in photocopying units to produce diverse products, which are known to have

adverse health effects. For example, formaldehyde is a Group 1 carcinogen in a 2004

International Agency for Research on Cancer evaluation (Cogliano et al., 2005).

Acrolein is an irritant and carcinogen (California Office of Environmental Health Hazard

Assessment, 2006). Peroxyactyl nitrate is a known eye irritant, as are some of the

products of ozone / terpene and ozone / isoprene chemistry (Kleno and Wolkoff, 2004;

Nojgaard et al., 2005). Matura et al. (2003 and 2005) suggested that

hydroperoxides formed through the oxidation of terpenes and terpenoids can be potent

contact allergens.

2.3.3 Volatile Organic Compounds

Barro et al. (2009) and Durmusoglu et al. (2010) opined that VOCs might cause

adverse health effects such as cancer even at very low concentrations. The key signs or

symptoms associated with exposure to VOCs include eye, nose, throat, skin irritations,

nose and throat discomfort, headache, allergic skin reactions, nausea, fatigue or

dizziness and shortness of breath. VOCs have also been linked with sick building

syndrome.

Many VOCs produce negative health effects if humans are exposed to high

concentrations. Long-term exposure to low concentrations of VOCs at or above

regulatory standards were found to result in liver and kidney damage and changes in

lipid metabolism as observed by Tanyanont and Vichit-Vadakan (2012).. Formaldehyde,

acetaldehyde and benzene are also suspected to be carcinogenic (Dutta et al., 2009).

Tsai et al. (2010) noted increased cardiovascular risks due to exposure to

propane, iso-butane and benzene. In addition, occupational VOC exposures have been

associated with decreased heart rate variability and increased inflammatory blood

markers (Ma et al., 2010).

Delfino et al. (2003) reported increased respiratory symptoms with increasing

outdoor levels of benzene, m-xylene, p-xylene, ethyl benzene and tetrachloro

ethylene. But, they did not observe a strong association between benzene exposure

and lung function. Weichenthal et al. (2012) also did not identify important

associations between total VOC exposures on cardio-respiratory outcomes.

2.3.4 Semi Volatile Organic Compounds

Xu and Zhang (2011) reported that alkyl phenols interfere with or block

hormones. Organochlorines might cause neurotoxicity and cancer. They might have

adverse effects on developing reproductive systems and on lactation.

Organophosphorus compounds have been reported to have multiple effects on

neurodevelopment and growth in developing tissue. They have been related to

respiratory disease in children through dysregulation of the autonomic nervous system.

Phthalates have been shown to have adverse effects on the development of male

reproductive tract, seemed to cause prenatal mortality, reduce intrauterine growth and

birth weight and were related to asthma and allergies in children. Poly brominated

diphenyl ethers caused impaired brain and nerve development, permanent learning and

memory impairment, behavioural changes, delayed puberty onset, foetal malformations

and thyroid hormone disruption. Polychlorinated biphenyls are classified under

developmental neurotoxicants. They have been shown to have harmful effects on

immune, reproductive, nervous and endocrine systems and cancer. Exposure to

polycyclic aromatic hydrocarbons might lead to high incidence of cataracts, kidney and

liver damage, increased risk of skin, lung, bladder and gastrointestinal cancers.

2.3.5 Toners

Gminski et al. (2011) recommended that exposure to toner might occur by direct

skin or eye contact, inhalation or ingestion. Balakrishnan and Das (2010) reported that

toner dust might irritate the respiratory tract, resulting in coughing and sneezing.

Symptoms such as non-allergic rhinitis, sore throat, asthma and pseudo allergic

inflammation of the respiratory tract, irritation of the skin and eyes, headache and sick

building syndrome seemed to be linked to toner emissions by many authors over the

years (Gminski and Mersch-Sundermann, 2006 and Jaakkola et al., 2007).

Inhalation of toner powder has been associated with sarcoidosis by Rybicki et al.

(2004), siderosilicosis by Gallardo et al. (1994) and granulomatous pneumonitis by

Ambruster et al. (1996b). Theegarten et al. (2010) reported the development of sub-

mesothelial deposition of carbon nanoparticles in the peritoneum of a photocopier

exposed worker.

Kitamura et al. (2009) observed a significant partial influence of toner exposure

on pulmonary function in the indices for peripheral respiratory tracts. However,

Nakadate et al. (2006) and Terunuma et al. (2009) observed no relationship between

toner-powder exposure through inhalation and the risk of adverse respiratory effects.

Experimental studies by Bai et al. (2010) found that exposure to toner particles

inhibited the normal growth of the mice and induced significant inflammatory responses

and lesions in the lung tissues. Morimoto et al. (2005) and Slesinski and Turnbull (2008)

reported that exposure to ultrafine and fine toner-emitted particles led to increase in

inflammatory response.

According to Totsuka et al. (2009), carbon black particles, one of the important

constituents of toners, are known to be genotoxic in in vitro and in vivo assay

systems. Moller et al. (2004), Pott and Roller (2005) and Mohr et al. (2006)

concurred that intra-tracheal instillation of very high doses of toner powder produced

significantly increased lung tumour rates in rats. They observed that carcinogenicity did

not depend on the composition of the different powders, but rather on particle size or

particle volume and on the potential of small particles to be deposited on the

lung epithelial cells. However, Morimoto et al. (2005) found toners to be non-

carcinogenic.

2.3.6 Selenium

The trace element selenium is essential for life but can also be toxic in humans.

As components of proteins, enzymes and hormones, they regulate biological functions,

particularly redox balance, inflammatory and immune responses. Pathological disorders

such as infections, cancers, cardiovascular and neurological diseases may be seen

when their homeostasis is modified. Serum concentrations of selenium provide useful

information in the clinical categorization of deficiency and toxicity states (Arnaud et al.,

2008). Steinbrenner et al. (2011) considered the therapeutic window of selenium to be

narrow and suggested that adverse health effects might occur due to supra-nutritional

selenium intake even below the levels required for intoxication.

Au et al. (2005) reported dizziness, fatigue and irritation of mucous membranes

in people exposed to selenium in workplace air. In extreme cases, pulmonary oedema

and severe bronchitis have been reported. The exact exposure levels at which these

effects might occur is not known, but they become more likely with increasing amounts

of selenium and with increasing frequency of exposure.

Risher et al. (2003) felt that the primary target organ for selenium exposure in

humans is the lung. Lesser effects might be observed in other organs / organ systems.

Workers acutely exposed to high concentrations of elemental selenium dusts have

reported stomach pain and headaches, whereas workers briefly exposed to high levels

of selenium dioxide dust experienced respiratory symptoms such as pulmonary

oedema, bronchial symptoms, symptoms of asphyxiation and persistent bronchitis,

elevated pulse rates, lowered blood pressure, vomiting, nausea and irritability. Several

occupational studies regarding chronic exposure describe respiratory effects such as

irritation of the nose, respiratory tract and lungs, bronchial spasms and coughing.

According to Navas-Acien et al. (2008), selenium supplements were found to

increase the risk of diabetes and hypercholesterolemia whereas Steinbrenner et al.

(2011) suggested that increased plasma selenium levels might be both a consequence

and a cause of diabetes.

2.4 Indoor Air Pollutants and Pulmonary Function Air pollution has been associated with numerous adverse health outcomes such

as respiratory symptoms, cardiovascular events, hospital admissions and mortality. In

air pollution studies, lung function is of interest as an objective measure of respiratory

health and an early predictor of cardio-respiratory morbidity and mortality (Gotschi et al.,

2008).

There are several tests of lung function which are used in epidemiological

studies. Spirometry is the most frequently used. It is a reliable, simple, non-invasive,

safe and non-expensive procedure, requiring only modest skills. It enables health

professionals to make an objective measurement of airflow obstruction and assess the

degree to which it is reversible. However, they require co-operation from the subject and

sufficient technical experience to ensure that they are made in a standard way. Several

reference values have been published which take age, sex and height into account but

with rather large differences between them. It is therefore recommended that in

epidemiological studies, examinations be carried out also on control groups to avoid

total dependence on standard reference values (Soriano et al., 2009). The methodology

for obtaining forced expiratory manoeuvres and derived parameters have been

standardised by the American Thoracic Society and the European Respiratory Society

first separately and now also jointly (Cazzola et al., 2008). Table 2 gives the list

of lung function parameters assessed by spirometry.

Table 2

Lung function parameters assessed by spirometry

Parameter Description

Forced vital capacity

(FVC)

The volume of air that can be forcibly and

maximally exhaled out of the lungs

Forced expiratory volume

in 1 second (FEV1)

The volume of air that can be forcibly exhaled

from the lungs in the first second of a forced

expiratory manoeuvre

Forced expiratory volume

in 6 seconds (FEV6)

The amount of air exhaled with maximum effort in

the first six seconds

FEV1 / FVC FEV1 / FVC ratio

Peak expiratory flow

(PEF)

The maximum flow rate achieved during the

forced vital capacity manoeuvre

Forced expiratory flow

25% - 75% (F25–75)

The amount of air expelled from the lungs during

the middle half of the forced vital capacity test.

Forced Expiratory Flow

25 % (FEF25)

The amount of air that was forcibly expelled in the

first 25% of the total forced vital capacity test.

Forced Expiratory Flow

50% (FEF50)

The amount of air expelled from the lungs during

the first half (50%) of the forced vital capacity test

Forced Expiratory Flow

75% (FEF75)

The amount of air that was forcibly expelled in the

first 75% of the total forced vital capacity test

Peak Inspiratory Flow

(PIF)

The fastest flow rate achieved during inspiration

Maximal Ventilatory

Volume (MVV)

The total volume of air moved during rapid and full

breathing for 12 -15 seconds

Lung function steadily increases from birth until early adulthood, culminates in a

so-called plateau phase in the mid-twenties and thereafter decreases with age. FEV1 is

a marker of airway obstruction. Flow measures are markers of small-airway function.

Review of Literature

FVC value in comparison with predicted values is the most commonly examined index

in routine spirometry testing as an indicator of restrictive process (Gotschi et al., 2008

and Hess et al., 2012). Figure 5 shows a model flow volume loop obtained by

spirometry.

Figure 5

Flow volume loop obtained by spirometry

www.morgansci.com (2012)

COPD is characterised by physiological abnormalities, including airflow limitation,

abnormalities in gas exchange and lung hyperinflation, reduced functional capacity,

associated with multiple co-morbidities and markers of systemic inflammation (Cazzola

et al., 2008; Yohannes and Ershler, 2011). COPD comprises the pulmonary

emphysema (condition anatomically defined by obstruction and enlargement of the

pulmonary alveoli), the chronic bronchitis (characterized by chronic cough and

expectoration) and the disease of small airways (pathologic condition that involves the

small bronchioles). Bakke et al. (2011) defined COPD as a preventable and treatable

disease with some significant extra pulmonary effects and added that the airflow

limitation is usually progressive and associated with an abnormal inflammatory

response of the lung to noxious particles or gases.

Review of Literature

Chronic obstructive pulmonary disease (COPD) is the major respiratory non

communicable disease. Mehta et al. (2012) reported that it affected around 10% of the

adult population and predicted that it will be the third cause of death and disability in the

world by the year 2020. It has been estimated that approximately 15–20% of the

population burden of COPD is attributable to occupational exposures.

Persistent, low-level, systemic inflammation might play a significant pathogenic

role in COPD. Elevated circulating levels of white blood cells, C Reactive

Protein (CRP), interleukins 6 (IL-6) and 8 (IL-8), fibrinogen and tumour necrosis factor

alpha have been reported in patients with COPD by Agusti et al. (2008). The pulmonary

inflammation in COPD appeared to increase with disease progression and during

exacerbations (Barnes et al., 2006).

Provinciali et al. (2011), Barbu et al. (2011) and Steinvil et al. (2008) suggested

the cells and mediators involved in the amplified inflammatory reaction were responsible

for the local and systemic effects of inflammation. In order to explain the systemic

effects of inflammation, several mechanisms were proposed: the systemic spread of

inflammation mediators from pulmonary compartment, the inflammatory reaction to

tissue hypoxia, the reaction induced by bacterial product lipopolysaccharide during

exacerbations, cigarette smoking involvement, the pulmonary hyperinflation, the skeletal

muscle dysfunction and bone marrow involvement .

2.5 Biomarkers of Pulmonary Damage

Bakke et al. (2011) defined biomarker as a biological marker that is objectively

measured and evaluated as an indicator of normal biological processes, pathological

processes or pharmacological responses to a therapeutic intervention.

Maiese (2009) explained that the term “biomarker” can refer to any entity that

occurs in the body and can be measured to predict the diagnosis, onset or progression

of a disease process. The term ‘biomarker’ can apply to the assessment of specific

genes, proteins, products of cellular processes, a series of biological processes or even

the response of cells or tissues to therapeutic strategies. In fact, some biomarkers may

have the additional benefit to function as a surrogate marker to be used to predict

clinical outcome in some cases.

In epidemiological studies, biomarkers should be easy to obtain, not too costly

and of a relatively non-invasive character. From the long history of air pollution

research, various biomarkers linking air pollution exposure to its adverse effects in the

respiratory and cardiovascular systems have been identified in human studies. Li and

Nel (2011) opined that most of these biomarkers are associated with two major

toxicological response pathways, oxidative stress and inflammation.

2.5.1 Oxidative Stress Markers

Halliwell (2007) defined oxidative stress as a serious imbalance between reactive

species production and antioxidant defenses. A disturbance in the balance between

pro-oxidants and antioxidants in favour of the former, leading to oxidative damage,

gives rise to an increase in oxidative stress.

According to Khansari et al. (2009), reactive oxygen species (ROS) might be

produced from both endogenous and exogenous cellular substances. Potential

endogenous sources include mitochondria, cytochrome P450, peroxisomes and activated

inflammatory cells. Cell destruction also causes further free radical generation.

Additional endogenous sources of cellular reactive oxygen species might be

neutrophils, eosinophils and macrophages. In addition, intracellular formation of free

radicals can occur by environmental sources including ultraviolet light, ionizing radiation

and pollutants such as paraquat and ozone. All these sources of free radicals might

have the potential to inflict oxidative damage on a wide range of biological

macromolecules. Figure 6 shows a schematic representation of oxidative stress caused

by air pollution.

Figure 6

Oxidative stress caused by air pollution

Review of Literature

8-isoPGF2 - 8-isoprostane (Delfino et al., 2011)

MDA - Malondialdehyde

oxLDL - Oxidised low density lipoprotein

GSH - Glutathione

Persistent oxidative stress may give rise to pathological conditions and is

increasingly implicated as a contributing factor to several human pathologies (Hulbert et

al., 2007).

Pinchuk et al. (2012) insisted that to relate to the development of oxidative

damage, we need at least one marker of oxidative damage. The most sensitive

biomarkers are lipids. Hence, lipid peroxidation is likely to reflect the balance

between pro-oxidative and anti-oxidative component systems. Moller and Loft (2010)

stated that the biomarkers of lipid peroxidation products included conjugated

dienes, lipid hydroperoxides, malondialdehyde, thiobarbituric acid reactive substances

and F2-isoprostanes measured in exhaled breath condensate (EBC), plasma, serum or

urine.

As stated by Lin and Thomas (2010) serum malondialdehyde a product of

cell membrane lipid peroxidation, is a marker of oxidative stress. 8-

Isoprostane is considered as a reliable oxidative stress marker in biological fluids.

Isoprostanes are prostaglandin-like compounds which are derived from the free radical-

catalyzed peroxidation of arachidonic acid on plasma membrane phospholipids (Barreto

et al., 2009).

Niki (2010) defined an antioxidant as a substance that, when present at low

concentrations compared to those of the oxidizable substrate, significantly delays, or

inhibits, oxidation of that substrate. Some antioxidants are proteins and enzymes, while

others are small molecules. Based on the mechanistic functions, four defense

mechanisms have been proposed. The antioxidants may be classified into preventing

antioxidants, scavenging antioxidants, repair and de novo antioxidants.

First line of defense: The preventing antioxidants function by suppressing the

formation of reactive oxygen and nitrogen species (ROS / RNS) for example,

reducing hydrogen peroxide and lipid hydroperoxides to water and lipid hydroxides,

respectively, or by sequestering metal ions such as iron and copper.

Second line of defense: The scavenging antioxidants remove active species rapidly

before they attack essential molecules. Superoxide dismutase converts superoxide

to hydrogen peroxide, while carotenoids scavenge singlet oxygen either physically or

chemically. Many phenolic compounds and aromatic amines act as free radical-

scavenging antioxidants.

Third line of defense: Various enzymes function by repairing damages, clearing the

wastes and reconstituting the lost function.

Fourth line of defense: The adaptation mechanism, in which appropriate antioxidants

are generated at the right time and transferred to the right position in right

concentration.

Some antioxidants might act as cellular signalling messengers to regulate the

level of antioxidant compounds and enzymes.

Total antioxidant capacity measures the capacity of free radical scavenging by

the radical scavenging antioxidants contained in the samples. The Trolox equivalent

antioxidant capacity assay is based on the reduction of the (2, 2’-Azino-di-[3-ethyl

benzthiazoline sulphonate]) ABTS radical cation in the presence of metmyoglobin,

activated by hydrogen peroxide. The results have been compared to those observed for

Trolox in similar conditions of concentration and time. The strong points of this assay

are that it can be applied for both water-soluble and lipid soluble antioxidants. It has

been adapted for clinical studies (Pinchuk et al., 2012).

2.5.2 Inflammatory Markers

Gabay (2006) pointed out that inflammation is a complex defense mechanism in which

leukocytes migrate from the vasculature into damaged tissues to destroy the agents that

can potentially cause tissue injury. Acute inflammation is a limited beneficial response,

particularly during infectious challenge, whereas chronic inflammation is a persistent

phenomenon that can lead to tissue damage. One hallmark of acute inflammation is that

initially the leukocyte infiltrate is mostly neutrophilic, but after 24 to 48 hours monocytic

cells predominate. In contrast, chronic inflammation is histologically associated with the

presence of mononuclear cells, such as macrophages and lymphocytes.

Review of Literature

Inflammation is a hallmark of the pathophysiology cascade leading to respiratory

diseases, such as asthma or chronic obstructive pulmonary disease (COPD). It is

important to understand the biological mechanisms and biomarkers of inflammatory and

oxidative stress pathways.

2.5.2.1 Leukotriene B4

Arachidonic acid is a polyunsaturated essential fatty acid that is converted by

cyclooxygenase and lipoxygenase to various biologically active derivatives, including

lipid mediators, also known as eicosanoids, such as prostaglandins, thromboxanes and

leukotrienes. These compounds were found to be responsible for pain and other

inflammatory symptoms. All the cells in the body except red blood cells synthesize

eicosanoids. Leukotriene B4 is the major arachidonic acid derivative produced through

5-lipoxygenase. It is found to promote migration of neutrophils to inflamed tissue

(Masclans et al., 2007 and Obied et al., 2012).

Leukotriene B4 (LTB4) is released by neutrophils and macrophages and plays a

regulatory role in the immune response to antigenic stimulation; it is a potent

chemotactic and chemokinetic agent for human polymorphonuclear leukocytes and a

margination factor for monocytes and macrophages. In addition, LTB4 was reported to

stimulate cytotoxic T cell, natural killer cell and suppressor T cell activity (Comandini et

al., 2009).

2.5.2.2 C Reactive Protein

C Reactive Protein (CRP) is a non specific acute phase reactant that is released

in the presence of inflammatory processes caused by various aetiologies. It is

synthesized in the liver and normally present in trace amounts in peripheral

circulation. In the presence of inflammation, CRP production is stimulated by systemic

cytokines (Dahdal and Dahdal, 2009). C Reactive Protein is reported to reflect

total systemic burden of inflammation in several disorders and has been shown to up

regulate the production of pro-inflammatory cytokines (Karadag et al., 2008)

Aronson et al. (2006) and Walter et al. (2008) felt that serum concentrations of

CRP might be associated with impaired lung function. Higher levels of systemic

inflammation might simply represent “spill over” from airways inflammation. CRP has

also been linked to increased risk of cardiovascular disease (Brook et al., 2009).

2.5.2.3 Interleukins

Interleukins are a group of pro-inflammatory cytokines (signalling molecules) that

are expressed by leukocytes. Their primary functions are to stimulate inflammatory cell

chemotaxis and to induce the maturation and proliferation of immune cells including

neutrophils, macrophages, T-helper cells, B-cells and natural killer cells (Lin and

Thomas, 2010).

According to Gabay (2006), interleukin (IL)-6 is produced at the site of

inflammation and plays a key role in the acute phase response. IL-6 was reported to

dictate the transition from acute to chonic inflammation by changing the nature of

leucocyte infiltrate from polymorphonuclear neutrophils to monocyte / macrophages. In

addition, IL-6 was found to exert stimulatory effects on T- and B-cells, thus favouring

chronic inflammatory responses. IL-6 has been shown to be a key player in

chronic inflammation and was found to be elevated in inflammatory diseases. Brook et

al. (2009) observed that increased concentrations of IL-6 were found to be associated

with an increased risk of cardiovascular events and mortality. IL-6 is directly involved in

regulation of the synthesis of CRP in the liver.

IL-8 is actively secreted in the extracellular space as a result of a variety of

cellular stimuli. A wide variety of cell types, including virtually all nucleated cells, are

potential sources of IL-8. However, the principal cellular sources of IL-8 are typically

monocytes and macrophages. Different stimuli including pro-inflammatory

cytokines, microbes and their products and environmental factors such as hypoxia were

suggested to induce production of IL-8. IL-8 was reported to bear the primary

responsibility for the recruitment and activation of monocytes and neutrophils, the

signature cells of acute inflammatory response. These functions suggest that IL-8 plays

a primary role in the various pathological conditions such as chronic inflammation

(Pourfarzam et al., 2009).

Apostolakis et al. (2009) reported that IL-8 might be highly sensitive to

oxidants. Anti-oxidants seemed to reduce IL-8 gene expression. The role of oxidants in

the regulation of IL-8 and other chemokines was proved to have relevance in the field of

cardiovascular disease. In addition, IL-8 was observed to have an important role

in the pathogenesis of chronic obstructive pulmonary disease (COPD), pulmonary

fibrosis and asthma. It was also suggested as a biomarker in diagnosis, activity, severity

or exacerbation of pulmonary diseases.

2.5.2.4 Eosinophil Cationic Protein

The Eosinophil Cationic Protein (ECP) is a small polypeptide that originates from

activated eosinophil granulocytes. A wide range of stimuli have been shown to induce

the secretion of ECP. It has many biological functions, including an immunoregulatory

function, the regulation of fibroblast activity and the induction of mucus secretion in the

airway. Oliveira et al. (2012) viewed that ECP is a potent cytotoxic molecule and has

the capacity to kill mammalian and non-mammalian cells. It was established to be a

member of the RNase A super family of proteins, involved in inflammatory processes

mediated by eosinophils (Comandini et al., 2009)

Bystrom et al. (2011) remarked that the majority of ECP is released after

the eosinophil has left the circulation. Several types of inflammatory stimulation have

been shown to cause eosinophil degranulation. Interaction with adhesion molecules,

stimulation by leukotriene B4, platelet activating factor, interleukin-5, immunoglobulins

and complement factors C5a and C3a all seemed to cause ECP release.

2.5.2.5 Myeloperoxidase

Myeloperoxidase (MPO) is a leukocyte-derived enzyme that catalyzes the

formation of a number of reactive oxidant species and impacts on nitric oxide through

complex mechanisms, including direct catalytic consumption resulting in endothelial

dysfunction (Tang et al., 2009). MPO is the most abundant component in the azurophilic

granules of leukocytes and it is found in neutrophils, monocytes and in some subtypes

of tissue macrophages. MPO is secreted upon leukocyte activation. It amplifies the

oxidative potential of H2O2 derived from leukocyte NADPH oxidases, xanthine oxidase,

uncoupled nitric oxide synthase and various isoenzymes by forming potent oxidants

capable of chlorinating and nitrating phenolic compounds. MPO-derived diffusible

radical species are capable of initiating lipid peroxidation as well as promoting an array

of post-translational modifications in target proteins, including halogenation, nitration

and oxidative cross-linking (Comandini et al., 2009).

Van der Veen et al. (2009) suggested that MPO is a heme-containing peroxidase

abundantly expressed in neutrophils and to a lesser extent in monocytes. Enzymatically

active MPO, together with hydrogen peroxide and chloride, produces the powerful

oxidant hypochlorous acid and is a key contributor to the oxygen-dependent

microbicidal activity of phagocytes. In addition, excessive generation of MPO-derived

oxidants has been linked to tissue damage in many diseases, especially those

characterized by acute or chronic inflammation. MPO was found to exert effects that

were beyond its oxidative properties. These properties of MPO were observed to be

independent of its catalytic activity affecting various processes involved in cell signalling

and cell–cell interactions and were capable of modulating inflammatory responses.

2.5.2.6 Intercellular Adhesion Molecule 1

Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin

superfamily. It is critical for the firm arrest and transmigration of leukocytes out of blood

vessels and into tissues. ICAM-1 is constitutively present on endothelial cells, but its

expression is increased by pro-inflammatory cytokines, in atherosclerotic tissues and

the progression of autoimmune diseases. The ligation of ICAM-1 on the surface of

endothelial or smooth muscle cells was observed to lead to the activation of several pro-

inflammatory signalling cascades. A circulating or soluble form of ICAM-1 (sICAM-1)

has been measured in various body fluids, with elevated levels being observed in

patients with atherosclerosis, heart failure, coronary artery disease and transplant

vasculopathy. sICAM-1 was reported to have signalling properties and found to invoke a

range of pro-inflammatory responses. It is produced by a variety of different

cells and is present in normal human serum at concentrations between 100 - 450 ng /

ml. sICAM-1 was found to bind competitively to ligands of membrane-bound ICAM-1

and involve in the progression of atherosclerosis and other chronic inflammatory

diseases (Lawson and Wolf, 2009).

2.5.2.7 Nitric oxide

Nitric oxide (NO) is an endogenous, soluble gas generated in the proximal and

lower airways and in the endothelium, where it exerts its main biological and patho-

physiological functions. Owing to its reactivity with hemoglobin and other biological

compounds, NO has a short lifetime in vivo. NO is an essential molecule in the

physiology of human body for nonadrenergic and noncholinergic neurotransmission,

vascular and nonvascular smooth muscle relaxation and protection against hyper

responsiveness. It also plays an important role in a variety of pulmonary functions,

including nonspecific but effective defense against pathogens, such as viruses, bacteria

and parasites, as well as against tumour cells and it is involved in the signalling

between macrophages and T cells. NO is able to interact with reactive oxygen species

(ROS) and to form other reactive nitrogen species (RNS), which are essential in many

physiological reactions and important for killing invading microorganisms. Enhanced

levels of ROS and RNS during oxidative stress in lung tissue which can be induced, for

instance, by infection or environmental pollutants might have a deleterious effect on

DNA, lipids, proteins and carbohydrates, thus leading to impaired cellular functions and

enhanced inflammatory reactions, necrosis and apoptosis (Corradi et al., 2011).

According to Bian et al. (2008) NO is a vasodilator. In addition, it mediates

several protective functions of the endothelium by inhibiting neutrophil activation and

adhesion, platelet adhesion and aggregation, vascular smooth muscle proliferation and

expression of pro-inflammatory cytokines. Thus, normal generation of nitric oxide might

prevent atherosclerosis development and its complications. In contrast to the beneficial

effects of NO at physiologic nanomolar concentrations, large amounts (micromolar)

might be toxic and pro-inflammatory.

Increased production of NO during inflammatory and immune processes affecting

the respiratory tract was suggested to cause respiratory tract injury thus contributing to

the pathophysiology of inflammatory airway diseases, as NO can react with superoxide

anion released from inflammatory cells, yielding the potent oxidant peroxynitrite.

Peroxynitrite adds a nitro group to tyrosine to produce the stable product nitrotyrosine.

NO also reacts directly with O2 to form nitrite, the oxidation of which, by neutrophil-

derived MPO or by other related peroxidases, yields nitryl chloride and nitrogen dioxide.

Although nitration of tyrosine is generally attributed to peroxynitrite, the peroxidase-

dependent nitrite oxidation pathway is also involved (Comandini et al., 2009).

The measurement of total nitrates and nitrites is often used as a surrogate

marker for nitric oxide, a molecule with multiple physiological functions, particularly

those related to vaso-relaxation (Bloomer et al., 2011).

2.5.2.8 Pneumoproteins

Haddam et al. (2009) suggested that over the past few years, novel approaches

have been developed to assess airway damage or inflammation caused by inhaled

pollutants non-invasively. Such an approach, referred to as pneumoproteinaemia,

consists of measuring plasma lung-specific proteins (pneumoproteins) reflecting the

cellular integrity or the permeability of the bronchoalveolar blood barrier. Among these

proteins, the most validated as a lung biomarker is the Clara cell protein (CC16, CC10

or secretoglobin 1A1), an anti-inflammatory protein secreted along the tracheobonchial

tree and the surfactant-associated protein D, which is secreted by the alveolar

epithelium in the deep lung. These proteins are reliable tools to detect airway damage

and to bring to light possible interactions between lung toxicants. Plasma levels of clara

cell protein and surfactant protein D have been considered as lung specific markers,

since these proteins are secreted from the cells in the bronchial epithelium (Engstrom et

al., 2012).

Kropski et al. (2009), reported that the biological function of CC16 remains

incompletely understood, although it has been demonstrated to interact with multiple

components of the inflammatory and coagulation cascades. CC16 inhibits

phospholipase A2 activity in vitro and in vivo, suggesting that it plays a role in

attenuating inflammatory responses. CC16 has also been implicated in feedback

inhibition of interferon gamma signalling, as well as modulation of T helper 2

responses to pro-inflammatory stimuli. Furthermore, CC16 appears to be activated by

tissue transglutaminases including activated factor XIII and inhibits thrombin-stimulated

platelet aggregation, suggesting a possible role in modulating the dysregulated

coagulation characteristic of acute lung injury. CC16 has been investigated as a

potential biomarker of lung epithelial injury in numerous disease states including

idiopathic pulmonary fibrosis, sarcoidosis, COPD, asthma, occupational or

environmental lung injury, bronchiolitis obliterans, chronic tobacco use and acute lung

injury.

Lomas et al. (2008) suggested that serum levels of CC16 largely reflect

protein produced by the lower respiratory tract and consequently has been suggested

as a marker of respiratory disease. CC16 acts as an immunosuppressant and provides

protection against oxidative stress and carcinogenesis. Serum levels were observed to

rise following acute exposure to smoke, chlorine, lipopolysaccharide and ozone. The

serum level of CC16 was found to be low in obliterative bronchiolitis, asthma, COPD

and in smokers.

2.5.4 Selenium and Selenoproteins

Selenium is an essential trace element involved in defense against oxidative

stress through selenium-dependent glutathione peroxidases and other selenoproteins.

In addition, selenium might have anti-carcinogenic effects not related to its antioxidant

properties (Blankenberg et al., 2003).

Burk et al. (2006) and Combs et al. (2011) viewed that three biomarkers in

plasma are used to assess selenium status and to predict toxicity. Two of them are

plasma selenoproteins (selenoprotein P and glutathione peroxidase-3). Plasma levels of

these selenoproteins are used primarily as nutritional biomarkers of the element. Their

concentrations are depressed in selenium deficiency but increased with increasing

selenium supplementation until reaching plateaus at levels determined by genetic and

environmental factors. The third biomarker is plasma selenium. It consists of selenium

in the forms of selenocysteine in the two selenoproteins and selenomethionine present

at methionine positions in all proteins plus small-molecule forms that contribute <3% of

the total.

Mammalian thioredoxin reductases are selenium-containing flavoprotein

oxidoreductases, dependent upon a selenocysteine residue for reduction of the active

site disulfide in thioredoxins. Along with its substrate thioredoxin and nicotinamide

adenine dinucleotide (NADPH), thioredoxin reductases form an essential system for

cellular redox regulation, cell proliferation and antioxidant defense. Their activity is

required for normal thioredoxin function (Arnér, 2009).

On the other hand, Conterato et al. (2011) believed that the easy access of

different compounds to the selenocysteine residue might make thioredoxin reductase a

potential target for inhibition by metals and electrophilic agents, which might disrupt its

function and promote oxidative events.

2.5.5 Genotoxicity Assessment

Genotoxicity is defined as any toxic modification of the structure or function of the

genetic material, DNA. An alteration in any part of the DNA that results in permanent

changes in cell function is called a mutation. The agents that cause such mutations are

known as genotoxins. It is usually performed on human peripheral lymphocytes (Peters

et al., 2009).

According to Valverde and Rojas (2009), biomonitoring of human populations

exposed to potential mutagens or carcinogens can provide an early detection system for

the initiation of cell dysregulation in the development of cancer. In recent years, the

comet assay, also known as a ‘‘single cell gel’’ electrophoresis assay, has become an

important tool for assessing DNA damage in exposed populations. This is the method of

choice for population-based studies of environmental and occupational exposure to air

pollutants, metals, pesticides, radiation and other xenobiotics.

2.6 Metabolomics as a Tool to Assess Pulmonary Function

Manney et al. (2012) opined that direct evidence for changes in inflammatory

state and oxidative stress in the lung in many individuals at multiple locations and times

in epidemiological studies is logistically challenging and hence scarce. However,

assessment of airway inflammation is now relatively easy using non-invasive means

such as induced sputum or exhaled breath condensate (EBC), although neither has

been assessed in epidemiological studies. Of the two, EBC collection is theoretically

easier to undertake in field studies.

Antus et al. (2010) and Loukides et al. (2011) felt that EBC collection represents

a rather appealing method that can be used to conveniently and noninvasively collect a

wide range of volatile and non-volatile molecules from the respiratory tract, without

affecting airway function, inflammation or the underlying disease process and can be

repeated within a short period of time.

EBC is collected by cooling exhaled air during spontaneous breathing. EBC

consists of water and a large number of slightly volatile (e.g. nitric oxide, carbon

monoxide, hydrocarbons) and non-volatile compounds (e.g. cytokines, lipids,

adenosine, histamine) exhaled in bio aerosol form. EBC contains a wide range of

mediators of inflammation (leukotrienes, prostaglandins, cytokines), oxidative stress

(hydrogen peroxide, lipid peroxidation markers) and nitrosative stress (nitrite, nitrate)

which could be useful to assess biomarkers of lung damage (Corradi et al., 2010). Davis

and Hunt (2012) felt that the principal physiological contributor to EBC is the variable-

sized particles or droplets from the airway lining fluid.

The metabolomic analysis of EBC is a simple approach for the study of

respiratory system diseases (Izquierdo-Garcia et al., 2011). Ala-Korpela et al. (2011)

defined metabolomics as an omics approach to identify and monitor metabolic

characteristics, changes and phenotypes with respect to various synergetic factors such

as environment, life style, diet and potential patho-physiological processes. It takes a

non-selective approach and enables the "metabolic fingerprint" of a sample to be

obtained. According to Baraldi et al. (2009), metabolomic analysis can be applied to the

study of biological fluids collected in non-invasive or minimally-invasive ways (e.g. urine,

exhaled breath condensate, blood). Sofia et al. (2011) observed that changes in

metabolite concentrations may reveal the range of biochemical effects induced by a

disease condition or its therapeutic intervention.

Different approaches can be applied in the study of metabolites. Metabolite

targeting is the most direct approach, aiming to identify and quantify a specific

metabolite, the product of an enzymatic pathway or the degradation product of a drug or

toxic agent. Metabolite fingerprinting, however, is a truly comprehensive methodology

that aims to classify samples without any apriori hypothesis to identify metabolite

patterns associated with a given pathological condition, with an environmental or

genetic change, or with exposure to a given toxic agent or drug. Metabolic fingerprinting

does not necessarily involve identifying each metabolite, but tries to detect the

metabolic characteristics that discriminate between groups of subjects. The search for

these metabolic patterns is open to new findings (Carraro et al., 2010).

The methods used in metabolomic analysis are generally based on mass

spectrometry (MS) or nuclear magnetic resonance (NMR)-based spectroscopy, since

these techniques can handle complex biological samples with a high sensitivity,

selectivity and throughput. MS is a powerful method for identifying and quantifying

metabolites and it is considered more sensitive than 1H-NMR. MS works in the range of

femtomoles while 1H-NMR works in the range of micromoles, if provided with cryoprobe.

1H-NMR spectroscopy enables the detection of almost all proton-containing metabolites

in a sample. Some of the advantages of this technique are that it is nonselective, fast,

nondestructive and usually demands no sample preparation. Recently, NMR has been

applied to biofluids to probe the metabolic status and to investigate different diseases

(Sofia et al., 2011).

NMR and MS spectra are highly complex and the biological information they

contain can only be extracted by applying bioinformatics tools, such as pattern

recognition methods. These are computer-based procedures that can be

classified as unsupervised or supervised. Once a metabolic pattern typical of a given

condition has been characterized, the analysis may go on to identify single

biomarkers relevant to sample clustering. Fundamental support for molecular

identification comes from various on-line databases, the most comprehensive of

which is the Human Metabolomic Database (Baraldi et al., 2009).