Human lung injury following exposure to humic substances and humic-like substances

Abstract

Among the myriad particles the human respiratory tract is exposed to, a significant number are distinctive in that they include humic substances (HS) and humic-like substances (HULIS) as organic components. HS are heterogeneous, amorphous, organic materials which are ubiquitous occurring in all terrestrial and aqueous environments. HULIS are a complex class of organic, macromolecular compounds initially extracted from atmospheric aerosol particles which share some features with HS including an aromatic, polyacidic nature. As a result of having a variety of oxygen-containing functional groups, both HS and HULIS complex metal cations, especially iron. Following particle uptake by cells resident in the lung, host iron will be sequestered by HS- and HULIS-containing particles initiating pathways of inflammation and subsequent fibrosis. It is proposed that 1) human exposures to HS and HULIS of respirable size (<10 μm diameter) are associated with inflammatory and fibrotic lung disease and 2) following retention of particles which include HS and HULIS, the mechanism of cell and tissue injury involves complexation of host iron. Human inflammatory and fibrotic lung injuries following HS and HULIS exposures may include coal workers’ pneumoconiosis, sarcoidosis, and idiopathic pulmonary fibrosis as well as diseases associated with cigarette smoking and exposures to emission and ambient air pollution particles.

Introduction

The human lung is regularly exposed to a wide variety of particles with a respirable diameter (<10 μm). Features of the clinical presentation and changes in human physiology and pathology following exposure to a large number of diverse particles appear to be comparable to some extent. These can include: 1) respiratory symptoms of cough, wheezing, and shortness of breath, 2) an acute, reversible decrement in pulmonary function and elevation in bronchial hyperreactivity, and 3) histopathological changes of acute inflammation and chronic parenchymal fibrosis. This shared clinical, physiological, and pathological presentation associated with exposure to disparate particles supports a common mechanism underlying their biological effects in the lung. Accordingly, a single pathway has been proposed through which the biological effects of all particles are generated (Ghio et al. 2016).

Inorganic particles including oxides and oxide minerals have oxygen-containing functional groups at their surface such as silanols (-Si-OH) in silica and silicate particles. The inhalation of particles with such surfaces comprised of oxygen-containing functional groups introduces an electronegative interface following their deprotonation at physiologic pH values. Among the cellular cations available for complexation by the particle surface, iron is kinetically favored as a result of its electropositivity, high affinity for oxygen-containing functional groups, and relative abundance (Dugger 1964). Following endocytosis (i.e. uptake) of an inorganic particle, surface functional groups react with intracellular iron to produce a coordination complex resulting in a functional deficiency of the metal within the cell (Ghio et al. 2004).

The complexation of cell iron is also relevant to particulate matter (PM) containing organic compounds (e.g. coal dust, soil particles, cigarette smoke particles, and emission and ambient air pollution particles). Surface functional groups in this PM can include alcohol, diol, epoxide, ether, aldehyde, ketone, carboxylate, ester, phenol, and catechol groups. Following exposure, organic particles also complex metal available in cells and tissues via these surface functional groups (Ghio et al. 1994; Ghio et al. 1996; Ghio et al. 2015; Ghio et al. 2016).

In the respiratory tract, particles have consistently demonstrated a capacity to accumulate iron from available cell sources reflecting the ability of the surface to complex host iron (Koerten et al. 1986). This response can be observed in the lungs of individuals exposed to PM containing organic compounds including coal (Figure 1), cigarette smoke, and woodsmoke particles (e.g. burning of biomass for cooking and heating in developing countries and use of wood burning stoves in developed countries) (Ghio et al. 1994). In response to the cell’s loss of essential metal to the particle, iron responsive protein is activated and metal import, such as that mediated by transferrin receptor and divalent metal transporter 1, is elevated to meet requirements (Ghio et al. 2013). Comparable to exposure to other compounds with a capacity to chelate cell iron, the response to the intracellular iron deficiency associated with particle exposure will include: oxidative stress, cell signaling (i.e. communication processes coordinating cell actions), activation of transcription factors (i.e. proteins initiating and regulating gene transcription), and release of pro-inflammatory mediators prior to apoptosis (i.e. programmed cell death) (Hileti et al. 1995; Laughton et al. 1989; Tanji et al. 2001; Kim et al. 2002; Lee et al. 2006; Huang et al. 2007; Markel et al. 2007; Liu et al. 2014; Zhang et al. 2014). This eventually culminates in the development of tissue inflammation and fibrosis (Ghio et al. 2013; Ghio et al. 2016).

Figure 1.

Stain of lung tissue collected at autopsy from a coal miner demonstrates an association between retained particles and accumulated iron (Gomori’s Prussian blue reaction; magnification approximates 400). Iron is blue with the lung tissue stained red. While normal human lung tissue does not stain for iron, here the metal is abundant.

Among the myriad particles the human respiratory tract is exposed to, a significant number are distinctive in that they include humic substances (HS) and humic-like substances (HULIS) as organic components. It is proposed that 1) human exposures to HS and HULIS of respirable size (<10 μm diameter) are associated with inflammatory and fibrotic lung disease and 2) following retention of particles which include HS and HULIS, the mechanism of cell and tissue injury involves complexation of host iron.

Humic substances and humic-like substances

HS are ubiquitous, heterogeneous, amorphous, organic materials occurring in all terrestrial and aqueous environments including composts, sediments, peat bogs, coals, rivers, lakes, and oceans (Stevenson 1985). The source and structure of HS are disputed. It has been postulated that HS originate from microbial degradation of dead plant matter. Specifically, it is thought that HS are the breakdown product of polysaccharides common to many living organisms (e.g. xylose, arabinose, and fructose from dead plants). In this process, polysaccharides are hydrolyzed to furfural which is oxidized to 4-oxo-2-butenoic acid and this compound polymerizes to HS.

HS include three different fractions: humic acid, fulvic acid, and humin. The humic acid fraction is not soluble in water under acidic conditions (pH<2) but soluble at higher pH values. Humic acid is the major extractable component of soil HS; it is dark brown to black in color. Fulvic acid is that fraction of HS soluble in water under all pH conditions and remains in solution after removal of humic acid by acidification; it is light yellow to yellow-brown in color. Humin is the fraction of HS that is not soluble in water at any pH value and is black in color.

HULIS are complex, organic, macromolecular compounds initially extracted from atmospheric aerosol particles and isolated from fog and cloud water (Graber & Rudich 2006; Zheng et al. 2013). As their presence in the atmosphere has been recognized only recently, knowledge of the sources of HULIS is deficient. Potential sources are considered to include wind erosion, biomass burning, and direct formation in the troposphere (Brigante et al 2008). These share some features with HS including an aromatic, polyacidic nature but differ in having a smaller average molecular weight and lower aromaticity (Graber & Rudich 2006). HULIS have been isolated from PM in cigarette smoke and combustion products including wood smoke particle, diesel exhaust particle, and ambient air pollution particle (Ghio et al. 1994; Ghio et al. 1996). Relative to HS in natural organic matter, HULIS from combustion products can be lower in oxygen content which is consistent with a loss of oxygen-containing functional groups by heating HS (Inshiwatar 1985; Perdue 1985). By weight, approximately 7–10% of tobacco smoke condensate, 8% of wood smoke particle, 5% of diesel exhaust particle, and 3% of ambient air pollution PM can be characterized as HULIS (Stedman et al. 1966; Ghio et al. 1994; Ghio et al. 1996).

As a result of having a variety of oxygen-containing functional groups (e.g. phenolic, carboxylate, keto, keto-enol, and carbonyl groups), both HS and HULIS complex metal cations (Ghio et al. 1996; Yang & Van den Berg 2009; Yamamoto et al. 2010; Town et al. 2012) (Figure 2). Through binding of metal cations, HS facilitate their mobilization, transportation, and accumulation in soils and waters (Prakash 1968). The high content of oxygen-containing functional groups in HS and HULIS favors the formation of stable complexes with metals but the sorption ability of iron is greatest among all of them (Erdogan et al. 2007). Accordingly, HS increase iron availability both in terrestrial and marine environments and increase productivity of the ecosystem (Prakash 1968).

Figure 2.

A postulated structure for HS and HULIS. These are complex, aromatic materials with numerous carboxylate and phenolic groups. Several potential sites for metal complexation by the substance are marked.

Comparable to all PM, HS- and HULIS-containing particles are taken up by cells resident in the lower respiratory tract. Once intracellular, host iron is sequestered by the particle as a result of its capacity to bind the metal. The intratracheal instillation of HULIS into the lungs of an animal model is associated with an accumulation of iron; much of this accumulation appears to be intracellular (Ghio et al. 1994). The sequestration of host iron and the associated cell metal deficiency initiates pathways leading to inflammation and fibrosis (Gau et al. 2001; Hseu et al. 2002; Cheng et al. 2003; Yang et al. 2004; Hseu et al. 2009; van Eijl et al. 2011; Hseu et al. 2014; Ghio et al. 2015).

Coal and lung disease

Coal contains natural organic matter. During the extraction of coal, deposition of this particle in miners’ lungs can result in a diffuse inflammatory and fibrotic interstitial lung disease recognized as coal workers’ pneumoconiosis (Morgan & Lapp 1976) (Figure 3). Human lung disease after coal dust exposure can also include evidence of chronic obstructive pulmonary disease (i.e. some combination of bronchitis and emphysema). The prevalence of lung disease among miners can increase with coal rank (Bennett et al. 1979). Increasing carbon content is one parameter that typically correlates with and determines coal rank. Accordingly, biological effect (e.g. inflammatory and fibrotic lung disease) can be associated with the content of HS in coal. Transition metals, especially iron, are concentrated in the lungs of miners with coal workers’ pneumoconiosis (Guest 1978). This accumulation of iron results, in part, from its complexation by HS which comprises up to 30% of dust weight in certain coals (Ghio & Quigley 1994). The association between concentrations of iron and hydroxyproline concentrations in the lung, reflecting collagen deposition and fibrosis, exceeds that between the mass of coal dust and hydroxyproline in a group of autopsied lung samples from coal miners (Ghio & Quigley 1994). Therefore, the disruption in iron homeostasis by the HS included in coal dust is likely to participate in the inflammatory and fibrotic disease following exposure to this particle. In addition to HS, several inorganic components of coal (e.g., quartz and kaolinite) share the same capacity to complex iron at the particle surface and, consequently, may also contribute to the toxicity of these dusts.

Figure 3.

Human lung disease following exposure to coal. Investigation supports coal workers’ pneumoconiosis to be a lung disease associated with HS in coal. The major risk factor for this diffuse inflammatory and fibrotic lung disease is coal mining (left). Coal workers’ pneumoconiosis frequently presents with radiologic evidence of opacities involving the upper lung fields (middle). A chest X-ray provides one single image of the entire chest with all structures included. Here, the lung between the ribs (thick arrows) shows small, rounded opacities throughout (thin arrows) reflecting an inflammatory and fibrotic injury after inhalation of coal dust. Staining and microscopic examination of the lung tissue reveals an inflammatory and fibrotic reaction which is initiated around the respiratory bronchiole where the particle is retained (right).

The relevance of iron coordination by HS in biological effect and lung disease after exposure to coal dusts of respirable size (<10 μm diameter) can assist in understanding certain clinical features of coal workers’ pneumoconiosis. The capacity of HS to adsorb host iron, following particle deposition and retention, predicts a potential to continue complexation of body sources of iron with worsening of disease even after the removal of the individual from the mine (i.e., clinical and radiographic progression of coal workers’ pneumoconiosis).

Soil and human lung injury

Soil includes mineral particles, water, air, and organic matter; HS comprise the majority of the organic matter. During agricultural activities and other acts requiring disruption of the soil, material is aerosolized. Consequently, exposure to soil particles has been and will continue to be ubiquitous throughout all societies. While almost every particle has been reported to impact human health and even exposure to the most benign PM has been documented to cause lung inflammation and fibrosis (e.g. pneumoconiosis following manipulation of iron oxides and carbon black), soil appears to be an exception to the relationship between particle exposure and human lung injury (Billings & Howard 1993; Szozda 1996). There is no report of human morbidity and mortality following the inhalation of soil particles despite millennia of continual manipulation of the ground. Among the possible reasons for a lack of recognition of specific lung disease after soil exposure are 1) misdiagnosis of such disease as idiopathic lung disease including sarcoidosis and idiopathic pulmonary fibrosis and 2) prior lack of recognition of a potentially responsible component. It is proposed that a number of sarcoidosis and idiopathic pulmonary fibrosis cases are caused by exposure to HS- and HULIS-containing particles.

Sarcoidosis is a disorder in which multiple organ systems demonstrate granulomas, a pattern of response to infection, inflammation, or the presence of a foreign substance (Govender & Berman 2015) (Figure 4). It is the most common interstitial lung disease of unknown etiology with a prevalence of approximately 15 of every 100,000 people in North America (Vallyathan et al. 1984). The vast majority of patients present between 20 and 45 years of age. The lung is the most frequently involved organ in sarcoidosis. The disease has been thought to represent a response to an unidentified inhaled exposure. A significant proportion of individuals with sarcoidosis are asymptomatic (40 to 60%), but the chest radiograph is abnormal in over 90% of patients. The diagnosis of sarcoidosis is one of exclusion and is assigned only after known causes of similar-appearing disease have been eliminated.

Figure 4.

Human lung disease following exposure to soil. Sarcoidosis can be a lung disease associated with exposure to HS in soil (left). Comparable to coal workers’ pneumoconiosis, it regularly presents with radiologic abnormalities. The computed tomography (CT) scan of the lung provides multiple images of the lung (similar to “slicing a loaf of bread) with the front of the chest being at the top of the picture and the back being at the bottom; between the two lungs are the heart and great vessels. In this image of a computed tomography scan of the lung from a sarcoidosis patient, there are numerous small, rounded opacities involving the upper lung fields (middle). Staining and microscopic examination of the lung tissue demonstrates granulomatous reactions, inflammation, and fibrosis as well as inclusion bodies (e.g. asteroid body in the photomicrograph) characteristic of sarcoidosis (right).

Early research demonstrated an association between sarcoidosis and exposure to soil (Gentry et al. 1955; Hurley et al. 1962). Agricultural employment with exposure to soil dust has been established as a risk factor for sarcoidosis (Kajdasz et al. 2001; Newman et al. 2004; Kreider et al. 2005; Taskar & Coultas 2008). Residence in rural areas and employment on a farm increase both exposure to soil and the risk for this specific lung disease (Gentry et al. 1955; Kajdasz et al. 2001). Epidemiological investigation has shown that the incidence of sarcoidosis increases as the geographic area moves away from the equator (Hosoda et al. 1997). Similarly, the content of organic matter in soil (which includes and correlates with HS) rises with distance from the equator (Lutzow et al. 2006). The higher rates of this disease in southeastern United States may reflect a higher concentration of organic matter in the soil and the importance of farming in this region (Gentry et al. 1955). In further support of a potential relationship between exposure to HS in soil and sarcoidosis, the risk for this disease can also be elevated with exposures to HULIS though the use of either wood stoves or fireplaces (Kajdasz et al. 2001). Extrapulmonary manifestations of sarcoidosis possibly reflect a tissue response to a lower molecular weight component which is water-soluble and subsequently systemically distributed (i.e. fulvic acid).

Pathologically, sarcoidosis is characterized by a number of non-specific cytoplasmic inclusions which can be microscopically identified within involved cells and tissues (Ma et al. 2007):

• Schaumann’s bodies, or conchoidal bodies, which occur in 48 to 88% of cases and consist of small calcifications with a lamellar morphology;

• calcium oxalate crystals;

• Asteroid bodies which are star-shaped spiculated structures usually observed in the multinucleated giant cells of sarcoidosis; and

• Hamazaki-Wesenberg bodies which are round, oval or spindle-shaped yellow-brown structures (1–15 micron) in lymph nodes.

It is unclear whether these inclusions are either a by-product of cell metabolism or exogenous material. Electron microscopy of humic acid can reveal a lamellar structure comparable to Schaumann’s or conchoidal bodies (Li et al. 2011). Calcium oxalate is present in many soils. Asteroid bodies can have the appearance of different soil constituents including calcium oxalate and pollen (Figure 4). Finally, yellow-brown structures in the lung can also support HS and HULIS exposure since components can range in colors which include yellow, orange, and brown. These inclusion bodies observed in tissues involved by sarcoidosis suggest exposure to HS, as well as other components of soil, and HULIS and therefore advocate an inhalation of PM originating from soil as a possible cause.

Idiopathic pulmonary fibrosis is a second lung disease associated with soil exposures. Idiopathic pulmonary fibrosis is a chronic fibrosing interstitial pneumonia of unknown cause limited to the lungs and associated with a biopsy showing a histologic pattern of usual interstitial pneumonia which includes varying quantities of alveolar inflammation and progressive fibrosis with the latter always predominating (American Thoracic Society 2000). The majority present between 40 and 80 years of age, though idiopathic pulmonary fibrosis may occur rarely in children. The diagnosis of idiopathic pulmonary fibrosis requires exclusion of other known causes of interstitial lung diseases. Agricultural workers suffer a greater rate of idiopathic pulmonary fibrosis (Baumgartner et al. 2000; Awadalla et al. 2012; Steele and Schwartz 2013). Farming is receiving increased attention for this disease since it may contribute to over 20% of all idiopathic pulmonary fibrosis (Taskar & Coultas 2008). The etiology has been suggested to result from inorganic components of soil dust (Schenker 2000; Schenker et al. 2009; Schenker 2010). This certainly is possible as there can be up to 2% silica in clay soils and up to 30% silica in sandy soils (Parks et al. 2003; Stopford & Stopford 1995). However, HS will also comprise significant quantities of these soils and the observed lung injury could be associated with natural organic matter. Finally, an etiologic role for HS in diffuse fibrotic disease among humans, such as idiopathic pulmonary fibrosis, is supported by observations of lung injury after aspiration of sediment (Noguchi et al. 1985; Mangge et al. 1993). These individuals, similarly exposed to HS but that which is included in sediment, demonstrate pulmonary fibrosis with aspirated foreign bodies including algae and pollens.

Cigarette smoking and lung disease

Some component of cigarette smoke condensate has a capacity to function as a ligand to bind transition metals comparable to HS in coal and soil (Qian & Eaton 1989). HULIS is associated with the incomplete combustion of tobacco leaf and can be isolated from cigarette smoke condensate (i.e. the particulate fraction of cigarette smoke) (Ghio et al. 1994). The concentration of iron increases in the lungs of smokers (McGowan et al. 1986). A brownish-black material with solubility properties and composition similar to HS can be isolated from the lungs of smokers (Ghio et al. 1994). Retention of this material in smokers’ lungs was associated with iron accumulation. Accordingly, it is possible that cigarette smoking introduces HULIS into the human lung where it has a capacity to complex essential metal and disrupt iron homeostasis.

Lung injuries after smoking are numerous and include interstitial lung disease (inflammatory and fibrotic) and cancer (Figure 5). These are proposed to follow exposure to HULIS in the cigarette smoke retained in the smoker’s lung with exposure to this material mediating a generation of oxidants, inflammation, and fibrosis. This is comparable to other models of inflammation and fibrosis which utilize compounds with a capacity to bind iron and alter its homeostasis (e.g. bleomycin) (Moore et al. 2013). Regarding fibrotic lung disease, cigarette smoking has been identified as a major risk factor for idiopathic pulmonary fibrosis with the odds ratio from various regions of the world ranging from 1.6 to 2.9 for the development of idiopathic pulmonary fibrosis in ever-smokers, that is current smokers and ex-smokers (Iwai et al. 1994; Hubbard et al. 1996; Baumgartner et al. 1997). The risk of developing idiopathic pulmonary fibrosis increases with the pack-years of smoking (Hubbard et al. 1996). Those with a history of smoking for 21 to 40 pack-years had an odds ratio of 2.3 (Baumgartner et al. 1997); the odds ratio for ever smoking was 1.6. Finally, tumor promoters and co-carcinogens are present in the weakly acidic (phenolic) fraction of cigarette smoke condensate, suggesting specific moieties of the HULIS as responsible components (Halliwell & Gutteridge 1981).

Figure 5.

Human lung disease following exposure to cigarette smoke particle. Epidemiologic investigation supports an association between 1) HS in soil and HULIS in cigarette smoke particle and 2) idiopathic pulmonary fibrosis (left). The computed tomography scan of the lung from a smoking patient shows irregular markings in the lower lung fields (middle) while staining and microscopic examination of the lung tissue reveals some combination of inflammation and fibrosis with the latter predominating (right).

Air pollutants and lung disease

A substantial mass fraction of tropospheric aerosols (up to 90%) can be comprised of natural organic matter (Jacobson 2000). This includes a mixture of aromatic, phenolic, and acidic functional groups that resembles HS (Dinar 2006; Graber & Rudich 2006). Combustion products of fuels are among the organic components in air pollution particles. An incomplete oxidation of several carbon-based materials can yield HULIS which are present in emission and ambient air pollution particles and confer a capacity to complex metals (Reemtsma et al. 2006). Quantification of total acidity and carboxylates demonstrate a presence of functional groups in these particles with a capacity to complex metals (Ghio et al. 1996). Evaluation by Fourier transform infrared spectroscopy exhibited a band consistent with C=O stretch of carboxylates (MacCarthy 1985). In addition, the Fourier transform infrared spectroscopy revealed a marked resemblance of material isolated from air pollution particle to HULIS isolated from cigarette smoke condensate (Ghio et al. 1994). Air pollution particles demonstrate measurable concentrations of transition metals with iron being that metal in greatest concentration (Ghio et al. 1994). The quantity of HULIS isolated from the air pollution particles correlates with the concentrations of included metal (Ghio et al. 1996). Exposure of respiratory epithelial cells to wood smoke particle initiated oxidative stress, cell signaling, transcription factor activation, and release of mediators relevant to an inflammatory injury (Ghio et al. 2015). Cell exposures to humic acid, used as a model for HULIS proposed as a component responsible for the biological effect of wood smoke particle, impacted the cells in an identical manner. Increased cell concentrations of available iron inhibited the pro-inflammatory response to both wood smoke particle and humic acid. The spectrum of lung disease following air pollution exposure is wide and includes inflammatory and fibrotic injuries; both are proposed to be associated with a functional deficiency of iron resulting after sequestration of host cell metal by the HULIS (Ghio et al. 2013). Finally, wood smoke increases the risk for idiopathic pulmonary fibrosis comparable to exposures to both soil and cigarette smoke (Scott et al. 1990; Iwai et al. 1994; Hubbard et al. 1996; Mullen et al. 1998; Baumgartner et al. 2000; Miyake et al. 2005; Gustafson et al. 2007; Kitamura et al. 2007; Tsuchiya et al. 2007; Taskar & Coultas 2008).

Conclusion

It is proposed that human exposure to particles which include HS and HULIS can be associated with inflammatory and fibrotic lung injuries including coal workers’ pneumoconiosis, sarcoidosis, and idiopathic pulmonary fibrosis as well as diseases after cigarette smoking and exposures to emission and ambient air pollutants. A weakness of the postulate is that disease proposed to result after inhalation of HS and HULIS, that is coal workers’ pneumoconiosis, idiopathic pulmonary fibrosis, and that following cigarette smoking and exposure to air pollutants, can also develop after other exposures (e.g. infectious agents and metals have been implicated in the etiology of sarcoidosis). It is reasonable to assume that specific cases of disease can be associated with exposure to HS and HULIS while other individuals with the same diagnosis may have a different cause. Another difficulty with the suggestion that HS and HULIS participate in these diseases is their dissimilar presentations (clinical, radiographic, and pathologic). However, the expression of disease following any exposure can be impacted by dose, clearance mechanisms, a myriad of other host factors, and chronicity of the exposure and the accompanying response. Chronic lung injury associated with sarcoidosis, idiopathic pulmonary fibrosis, and cigarette smoking can demonstrate identical presentations. Accordingly, distinct patterns of disease presentation may reflect interactions between the exposure and host factors and chronicity rather than discrete etiologies.

It is possible that HS and HULIS in coal, soil, cigarette smoke, and air pollution particles sequester host cell iron initiating molecular pathways of inflammation and fibrosis. Exposure to other xenobiotic agents with the equivalent capacity to coordinate metal cations impacts a comparable inflammation and fibrosis (Lovstad 1991; Ueda et al. 1993; Elias et al. 2002). Investigation defining associations of HS and HULIS with lung injury will likely necessitate exposures of living systems to these substances following treatments intended to increase tissue concentrations of available metal (e.g. inhaled iron compounds).