Diesel exhaust exposure, its multi-system effects, and the effect of new technology diesel exhaust

Environment International 114 (2018) 252–265

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Review article

Diesel exhaust exposure, its multi-system effects, and the effect of new technology diesel exhaust Haley Reisb,1, Cesar Reisa,b, ⁎⁎ Lawrence Beesonh,

T

⁎,1

, Akbar Sharipc, Wenes Reisa, Yong Zhaod,e,f, Ryan Sinclairg,

a

Department of Preventive Medicine, Loma Linda University Medical Center, 24785 Stewart Street, Suite 204, Loma Linda, CA 92354, USA Loma Linda University School of Medicine, 11175 Campus Street, Loma Linda, CA 92350, USA c Department of Occupational Medicine, Loma Linda University Medical Center, 328 East Commercial Road, Suite 101, San Bernardino, CA 92408, USA d School of Public Health and Management, Chongqing Medical University, Chongqing, China e Research Center for Medicine and Social Development, Chongqing Medical University, Chongqing, China f The Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing, China g Center for Community Resilience, School of Public Health, Loma Linda University, Loma Linda, CA 92350, USA h Center for Nutrition, Healthy Lifestyle, and Disease Prevention, School of Public Health, Loma Linda University, Loma Linda, CA 92350, USA b

A R T I C L E I N F O

A B S T R A C T

Handling Editor: Robert Letcher

Exposure to diesel exhaust (DE) from vehicles and industry is hazardous and affects proper function of organ systems. DE can interfere with normal physiology after acute and chronic exposure to particulate matter (PM). Exposure leads to potential systemic disease processes in the central nervous, visual, hematopoietic, respiratory, cardiovascular, and renal systems. In this review, we give an overview of the epidemiological evidence supporting the harmful effects of diesel exhaust, and the numerous animal studies conducted to investigate the specific pathophysiological mechanisms behind DE exposure. Additionally, this review includes a summary of studies that used biomarkers as an indication of biological plausibility, and also studies evaluating new technology diesel exhaust (NTDE) and its systemic effects. Lastly, this review includes new approaches to improving DE emissions, and emphasizes the importance of ongoing study in this field of environmental health.

Keywords: Diesel exhaust Diesel exhaust particles Air pollution Eyes Kidney Cardiovascular Respiratory Central nervous system Hematopoietic New technology diesel exhaust

1. Introduction Diesel exhaust (DE) is a complex mixture of hydrocarbons, gases, sulfur, and particulates produced during the combustion of diesel fuel (Hesterberg et al., 2011). It is considered an important source of ambient particulate matter (PM) in traffic-related air pollution. It has been estimated that 20–70% of PM is attributed to combustion-derived particles from traffic (Geller et al., 2005; Lanki et al., 2006), and up to 90% of PM in urban areas is traffic-related (Mazzarella et al., 2007). According to the study published by the Global Burden of Disease 2016 Risk Factors Collaborators, air pollution is among the leading top 10 risk factors for mortality in men and women in 2016 and has been considered a risk factor since 1990. In addition, occupational exposure from diesel exhaust engines was among the risks with an increase in its summary exposure value, a measure of exposure for each risk

(Collaborators, 2017). Moreover, according to the World Health Organization, > 7 million premature deaths annually are linked to air pollution, making it the largest single environmental health risk globally (WHO, 2014). In 2013, the International Agency for Research on Cancer (IARC) designated air pollution as a Group 1 carcinogen to humans based on the accumulating evidence regarding the relationship between particulate matter exposure and lung cancer risk (IARC, 2013). Other organ systems affected by the carcinogenic effects of DE include the central nervous system (CNS) and hematopoietic system (Danysh et al., 2015; Filippini et al., 2015). Non-carcinogenic changes are also associated with DE exposure, including retinal edema, conjunctivitis, bronchospasm, cough, systolic dysfunction, and changes in heart rate variability. Understanding the carcinogenic and systemic risks establishes the importance of reducing air pollution and improving the use of biomarkers to determine the level of exposure and its impact on

⁎

Correspondence to: C. Reis, Department of Preventive Medicine, Loma Linda University Medical Center, 24785 Stewart Street, Suite 204, Loma Linda, CA 92354, USA. Corresponding author. E-mail addresses: [email protected] (C. Reis), [email protected] (L. Beeson). 1 Authors contributed equally to this work. ⁎⁎

https://doi.org/10.1016/j.envint.2018.02.042 Received 5 September 2017; Received in revised form 24 February 2018; Accepted 24 February 2018 0160-4120/ © 2018 Elsevier Ltd. All rights reserved.

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Environment Protection Agency (EPA)- compliant heavy-duty diesel engines. The emissions were well below the 2007 standards, with only 13% of PM being elemental carbon, or soot. An 86% total reduction in PM emissions was found compared to a 2004-technology engine (Khalek et al., 2011). This review brings an important perspective on DE exposure. It takes on a multi-system approach in discussing DE and the consequences of both acute and chronic exposure. This review emphasizes the importance of identifying the specific components that make DE dangerous, studying NTDE and its epidemiological effects, and carefully evaluating potential alternative fuel options and whether they too pose a threat to biological processes.

Table 1 Traditional diesel exhaust (DE) and new technology diesel exhaust.

Carbon Elements with sulfur/sulfate Unburnt fuel Ash and other Unburnt fuel Elements without sulfur

TDE

NTDE

41% (primarily elemental carbon) 14% (sulfate and water) 25% 13% 7% 0%

13% elemental carbon + 30% organic carbon 57% 0% 0% 0% 4%

This table allows comparisons of major components between pre-2007 Diesel exhaust and post-2007 Diesel exhaust (Hesterberg et al., 2011). After institution of the NTDE the amount of carbon present in the exhaust has decreased drastically.

2. Methods We searched the PubMed database for literature on diesel exhaust and its multi-system effects, including epidemiology and pathogenic mechanisms. The systems included in this review were chosen based on the amount of literature discussing the organ system, and the overall burden of DE related to that organ system. Our systems were also based on the studies we found that measured biomarkers. Most of the biomarker studies focused on the respiratory and cardiovascular systems and are included in the respective sections. We also chose CNS and hematopoietic because DE is implicated in causing carcinogenic effects to these body systems. Finally, the visual and renal systems provided information on areas of human physiology that are not normally considered troubling upon exposure to DE, yet studies find pathogenesis in these system upon DE exposure. The inflammatory system, though not a section of its own, is incorporated into the pathogenesis of diseases related to DE exposure and is discussed throughout the review. Most of the organ system searches had a filter to include studies that were published within the last five years, except for the respiratory system (Fig. 1). This portion of the research included studies from within the last 10 years to fully elucidate the pathogenesis of DE on this organ system (Table 1). Most of the studies included in this review were published after the EPA standards were updated in 2007 to decrease DE emissions, and the term DE was used to describe the type of exhaust being studied. The specific components of the DE were not described, making it difficult to

cellular, oxidative, and inflammatory processes. The Environmental Protection Agency (EPA) placed standards in 2007 to protect against DE exposure and emissions. US EPA 2007/2010 Heavy-Duty Engine and Vehicle Standards and Highway Diesel Fuel Sulfur Control Requirements generated a movement towards developing enhanced technology to emit 90% less concentrated particulate constituents compared to traditional DE. The new standards are estimated to reduce PM emissions by 110,000 tons each year and toxic air pollutants such as benzene by 17,000 tons in the U.S. annually (MECA, 2017). The newly designed diesel engines and utilization of new technology diesel exhaust (NTDE) decreases the carcinogenic risk and reduces the physiological impact of particulate matter (Costantini et al., 2016). Animal studies investigate the effects of NTDE, specifically its effects on the respiratory and cardiovascular system, and compare these outcomes to former studies that used traditional diesel exhaust (TDE). According to preliminary toxicological data, significant chemical distinctions exist between NTDE particulate and DE particulate matter from pre-2007 diesel technology, including differences in biological responses. The presence of organic carbon and a higher percentage of elements with sulfur set NTDE apart from traditional DE (Table 1). Khalek et al., as part of the Advanced Collaborate Emissions Study (ACES), measured PM emissions and three other regulated emissions (carbon monoxide, non-methane hydrocarbon, and nitric oxide) on U.S.

Fig. 1. Schematic of search strategy. This figure includes our search strategy for diesel exhaust and its effects on multiple organ systems. After checking papers in English published over the last 5 years, we excluded reviews and commentaries and kept relevant papers.

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Table 2 Particulate matter (PM) defined. Name

Definition

Components

Sources

Authors

PM2.5

- PM2.5 is defined as fine particulate matter, with an aerodynamic diameter < 2.5 μm. - PM2.5 is considered “fine particles”

PM10 is the fraction of aerosol particles with aerodynamic diameter < 10 μm - PM10 is considered “inhalable particles”

Ultrafine particles (UFP)

Ultrafine particles are typically designated as those < 0.1 μm or with aerodynamic diameter of 100 nm

- Vehicles exhaust emissions - Resuspended crystal dust - Secondary sulfates - Industrial emissions - Vehicles exhaust emissions - Resuspended crystal dust - Secondary sulfates - Industrial emissions - Motor traffic - Indoor air in some facilities

(Fine et al., 2008; Sun et al., 2015)

PM10

- Organic carbon - Elemental carbon - Ammonium sulfate - Ammonium nitrate - Other inorganic constituents - Soil - Dust - Sea salt - Bioaerosols

- Organic carbon - Metals - Inorganic ions

(EPA, 2012), (Schiliro et al., 2015; Yun et al., 2015)

(Kumar et al., 2014; Li et al., 2003; Slezakova et al., 2017)

This table includes definitions of different forms of PM2.5, PM10, and Ultrafine Particles (UFP). It is important to understand that diesel exhaust contributes to all three forms of PM, and it is often used an indicator of pollution when conducting experimental animal studies and human studies, and when gathering data for epidemiological air pollution studies.

functional changes associated with neurotoxicity from pollution exposure. Deciphering the associations between PM exposure and childhood CNS tumors can be challenging, as direct exposure during childhood in addition to exposure in utero could affect the risk, in addition to length of exposure, and underlying genetic susceptibilities. A large population based study aimed to determine a relationship between maternal air pollution exposure and CNS tumors in their offspring. Using the Texas Cancer Registry from 2003 to 2009 and maternal residential information, they found that for every kilometer closer to a major highway, the odds of offspring having a CNS tumor increased by 30% (CI 1.0, 1.7) (Spycher, 2016). A pooled analysis of case-control studies of children < 15 years of age with a CNS tumor diagnosis showed different results. Using data from France, Germany, and the UK, researchers determined parental occupation at time of pregnancy to assess their exposure to various toxins, including polycyclic aromatic hydrocarbons (PAH) and diesel exhaust. Paternal exposure to toxins including PAH did show an increase risk for CNS tumors, but it was not statistically significant. No increased risks were found for maternal exposure to PAH and diesel exhaust (Huoi et al., 2014). Another studying conducted in Texas using the Texas Cancer Registry from 2001 to 2009 looked at CNS tumors in residents < 15 years of age and the association with ambient air pollution. Exposure grades were based on where the child was living at time of diagnosis, and exposure categories were based on statewide distributions of ambient 1,3-butadiene, benzene, and PM concentrations. The strongest associations were with astrocytoma incidence and 1,3-butadiene and PM, medulloblastoma incidence and PM, and primitive neuroectodermal tumors (PNET) incidence and 1,3-butadiene, benzene, and PM (Danysh et al., 2015). The association of PM with CNS tumors in children suggest an increased risk, but more quantitative study or meta-analysis using the accumulating evidence from cohort studies may elucidate the period in which the highest risk occurs.

discern if the emissions were TDE or NTDE. We include a section on recent animal studies using NTDE and their effects on inflammation and oxidative stress, as these were the only studies that specifically stated NTDE was being used in the experiments. Animal studies and human studies included in this review determined the level of air pollution by measuring the amount of PM study subjects are exposed to. Experimental studies discussed in this review described the type of PM study subjects were exposed to in the controlled environment and equated the exposures to non-experimental settings. The three types of PM discussed in the literature on DE include PM2.5, PM10, and ultrafine particulate matter (UFPM), as DE is a component of all three forms (Table 2).

3. Organ systems and consequences from diesel exhaust exposure 3.1. The central nervous system The BREATHE project aimed to evaluate the effects of air pollution exposure on neurobehavioral development in school aged children. Several articles that arose from the project found important effects of air pollution on the developing brain. Using school aged children in Barcelona, Spain, a study measured cognitive development for one year using validated computerized tests that measured working memory and attention functions. Elemental carbon, ultra-fine particulate matter (UFP), and NO2 were recorded both indoors and outdoors at the schools during the 12-month period and used in the analysis. At baseline, the difference in working memory between low- and high-exposure schools was 5.3 points, while after 1 year this difference had increased to 9.9 points, indicating a negative relationship between cognitive function and air pollution levels (Sunyer et al., 2015). To investigate anatomical and functional changes associated with air pollution, investigators used neuroimaging on a cohort of children in Barcelona with a mean age of 9.7. (Pujol et al., 2016). Though this study did not specifically determine the neurotoxic agents responsible for the changes seen on imaging, they used NO2 and carbon as pollution indicators, as 80% of NOx emissions in Barcelona arise from road traffic (Lus, 2012). Using multiple modalities of neuroimaging, including functional MRI, magnetic resonance spectroscopy, and diffuse tensor imaging, they found that vehicle exhaust-related pollution was associated with functional brain changes in integration and segregation capabilities associated with mental processes and stimulus-driven operations. There was no effect seen on imaging indicating changes in anatomy or metabolism (Pujol et al., 2016). Identifying these changes in functional connectivity in pre-adolescent children suggests prolonged exposure may impart a greater impact on the structural and

3.1.1. Pathogenesis Air pollution exposure is thought to stimulate oxidative stress and neuroinflammatory processes via microglial activation. Levesque et al. conducted an in vitro animal study and determined that ultrafine particles and diesel exhaust extracts activate microglia, cause elevated H2O2 production, and induce a Parkinson's disease-like pathology with impaired dopamine uptake (Levesque et al., 2013). Roque et al. used cerebellar granule neurons and microglia in an in-vitro study to understand the role of microglia and neuronal cell death. Microglia induce neuronal toxicity through neuroinflammatory mechanisms that increase the mRNA levels of pro-inflammatory cytokines including IL-6 and IL1-β (Roque et al., 2016) Allen et al. exposed mice to human254

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15 km outside of Sao Paulo who lived farther outside the city (Group 2). For every 50 μg/m3 increase of PM2.5 measured in the air, a statistically significant decrease in IL-5 and IL-10 levels was observed. These individuals had an adaptive ocular response to ambient air pollution with increased tearing and subsequent decrease in cytokine production (Matsuda et al., 2015). Torricelli et al. found a similar response in normal subjects exposed to ambient air pollution, with decreased tear osmolarity levels (Torricelli et al., 2014). The excessive tearing may generate a diluted state, leading to the decreased inflammatory and pro-inflammatory cytokines.

relevant levels of ultrafine concentrated ambient particles (200,000 particles/cm3) in the postnatal period to better understand the mechanisms leading to adverse effects in the CNS. In the female mice, TNF-α and IL-1 β were persistently increased, whereas IL-6 was decreased in both male and female mice, suggesting dysfunctional microglia, neuroinflammatory processes, and disruptions in synaptic plasticity. Lateral ventricle dilation was observed only in male mice exposed to postnatal air pollution, a pathological hallmark associated with poor neurodevelopmental outcome, autism, and schizophrenia (Allen et al., 2017). Though the results regarding IL-6 varied between animal studies, consistent increases in IL-1 β were noted, confirming a proinflammatory process. Bolton et al. found a greater long-term impact of in-utero air pollution exposure in male mice. They were found to have significant anxiety and memory deficits in adulthood associated with increased pro-inflammatory IL-1 and decreased anti-inflammatory IL-10, whereas female adult mice had increased IL-10, which likely served as a protective mechanism against prenatal neurotoxicity (Bolton et al., 2012). Another in-utero animal study found exposure to DE particles chronically activated the dorsal raphe nucleus, leading to increased serotonin levels and anxiety in 6-week old male offspring (Yokota et al., 2016). A recent study used a novel simulated vehicle exhaust exposure model that exposed rats to high dose brief exposure, and low dose prolonged exposure of vehicle exhaust for two weeks. Analysis was aimed at characterizing the effects of oxidative stress caused by the prooxidant components NO2, CO2, and carbon monoxide. The exposures of the prooxidant gases were expected to mimic someone living near highways for 1.5 years. While no change in body growth parameters were noted, an increase in anxiety-like behavior and impaired learning-memory function was observed in rats during both types of exposures. Authors suggest the prooxidant components induce molecular changes in brain regions of pre-frontal cortex, hippocampus, and amygdala (Salvi et al., 2017). To study the relationship between diesel exhaust particles and carcinogenicity and tumor-initiating capabilities, Misaki et al. used mouse embryonic fibroblast cells to study the effects of PAH and found them to be tumorigenic (≥10 foci following exposure to < 100 nM). The induction of tumorigenesis leads to dysfunction in aryl hydrocarbon receptor activation to oxidative stress, including antioxidant heme oxygenase-1 response activation (Misaki et al., 2016). Animal studies investigating the effects of DE on the CNS give biological plausibility to the associations found in epidemiological studies, but a specific look at CNS tumors after diesel exhaust exposure in utero and during development is a topic for future research.

3.2.1. Pathogenesis To better understand the pathogenesis behind DE exposure and allergic conjunctivitis, human conjunctival epithelial cells were studied in vitro. Liver and activation-regulated chemokine is able to recruit T-cells and dendritic cells via chemokine receptors and was detected in the epithelial cells after DE exposure, indicating an active inflammatory response (Fujishima et al., 2013). A recent study by Kim et al. studied the effect of acute exposure to DE product on rats for 1 h and compared to normal controls. They found that after acute exposure, the thickness of the retina was increased to 258 ± 96 μm in the diesel group compared to 113 ± 9 μm in the control group. Specifically, the inner plexiform, inner and outer nuclear, and the rod/cone cell layers showed statistically significant thickening (p < 0.01, 0.017, 0.004, 0.001, respectively). Capillary venous congestion was also noted in the group exposed to DE. Retinal thickening results in hypoxia-induced edema, leading to visual impairments and retinal damage (Kim et al., 2016). The investigation into air pollution and its acute and chronic effects on the visual system provide further evidence into the inflammatory and neovascular responses involved with exposure, and present context for future studies to develop strategies that reduce exposure, using cytokines and growth factors as a measure of effectiveness. 3.3. The hematopoietic system Though rare, leukemia is the most common malignancy affecting children < 15 years old, with the majority of the cases being acute lymphoblastic leukemia. In addition, the causes for leukemia remain largely unknown. A case control study conducted in Oklahoma used the Oklahoma Central Cancer Registry to identify cases of acute leukemia in children diagnosed before age 20. Using road density and NO2 concentrations, a marginal interaction was found between NO2 and urbanization for children with acute myelogenous leukemia after adjusting for maternal education (p = 0.09) (Janitz et al., 2016). Another study, by R-Nielsen et al. demonstrated an odds ratio for acute myelogenous leukemia of 1.20 (95% CI 1.04–1.38) per 20 μg/m3 increase in NOx and 1.31 (95% CI 1.02–1.68) per 10 μg/m3 increase in NO2. Evidence suggests an association between long term exposure to air pollution and acute myeloid leukemia in the general population (Raaschou-Nielsen et al., 2016). A meta-analysis in 2015 was performed to capture the epidemiological evidence on the risk of childhood leukemia following long-term exposure to traffic exhaust. Using 20 studies, they found that traffic air pollutants increase the risk of childhood leukemia. Their results found postnatal exposure to be more important than prenatal exposure in increasing the risk of acquiring childhood leukemia. Working towards reducing the release of contaminants from traffic, including benzene, may help reduce the incidence of childhood leukemia, particularly those with specific genetic susceptibility (Filippini et al., 2015). A census based nationwide cohort study in Switzerland focused on hematopoietic and lymphatic malignancies and their association with residence near a highway. Among children living < 500 m from a major highway, the risk for any type of cancer, was increased. Incidence rate ratios comparing children living < 100 m from a highway with unexposed children were 1.57 (95% CI 1.09–2.25) for leukemia and 1.64 (95% CI 1.10–2.43) for acute lymphoblastic leukemia. Associations

3.2. The visual system Using data collected from the BREATHE project, Dadvand et al. investigated the relationship between air pollution and the use of eyeglasses as a surrogate for myopia in school aged children. Exposure to NO2 and PM2.5 was measured and increased levels were associated with an increased likelihood of myopia. Specifically, cross-sectional analysis found an interquartile range increase in NO2 at home was associated with a 16% increase in eyeglasses (95% CI 3%–29%), and a 32% increase in eyeglasses with increased NO2 exposure at school (95% CI 9%–59%). The association of NO2 and myopia after performing a longitudinal analysis (3 years) became weaker and lost significance, and the authors believe more of the eyeglass use in the longitudinal analysis can be attributed to non-pollution related myopia compared to the cross-sectional analysis. In addition, the study did not consider the use of contact lenses, which could have biased the outcome towards null (Dadvand et al., 2017). Matsuda et al. studied air pollution and its effect on inflammatory tear cytokine levels using subjects who lived and worked in the city of Sao Paulo, Brazil and were chronically exposed to ambient air pollution (Group 1). They were compared to a cohort of individuals that worked 255

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were strongest for those diagnosed before age 5 living < 100 m from a highway (Spycher et al., 2015). This study determined an association between leukemias and proximity to highways, but it did not distinguish between the individual pollutants, a key area of research needed to advance efforts in diesel exhaust components. A population based cohort study by Malagoli et al. determined annual exposure to benzene and PM10 from traffic pollution to assess the risk for leukemias in urban areas. The odds ratio of leukemia associated with living in urbanized area was 1.4 (95% CI 0.8–2.4) and 1.33 (95% CI 0.8–2.0), regardless of exposure to benzene and PM10 from vehicular traffic. This study suggests an underlying susceptibility for leukemia in children living in urban areas due to insufficient microbial challenges and extremely hygienic conditions in early childhood. This results in an altered immune response to infection encountered later in life, while those living in rural areas are exposed to the outdoors and more microbial agents, thus generating adequate stimulation of the immune system (Malagoli et al., 2015). Other studies have generated different results compared to Malagoli et al. A case-control study conducted in France investigated the role of residential exposure to heavy-traffic roads, specifically benzene concentrations, in the occurrence of childhood leukemias. Estimated benzene concentrations greater than the median (1.3 μg/m3) was associated with acute myelogenous leukemia (odds ratio = 1.6, 95% CI: 1.0–2.4) but not with acute lymphoblastic leukemia. The increase in acute myelogenous leukemia incidence was associated with a high density of heavy-traffic roads within 150 m of a child's home (Houot et al., 2015). Studying and measuring exposure to specific components of air pollutants such as benzene or NO2 can be utilized to determine which components pose the greatest risk for childhood leukemia, and the underlying pathogenic mechanisms that lead to dysfunction in human hematopoietic system.

this study support the importance of reducing exposure, identifying the causative agents in PM2.5 that increase the risk for lung cancer, and improving air quality standards. A meta-analysis responded to the IARCs recent designation of air pollution as a Group 1 carcinogen and aimed to quantitatively analyze the evidence. Using 18 studies to perform their analysis, and restricting studies that posed potential confounders, they found the meta-relative risk for lung cancer associated with PM2.5 to be 1.09 (95% CI 1.04–1.14). When they analyzed data according to smoking status, former smokers had the greatest risk of lung cancer with PM2.5 [1.44 (95% CI 1.04–2.01)] (Hamra et al., 2014). It is important to note that most of the data collected retrospectively for epidemiological studies investigating lung cancer incidence and increase risk among those with high exposure to traffic-related air pollution was done in a time span that includes years preceding and proceeding the 2007 enactment of the U.S. EPA 2007/2010 Heavy-Duty Engine and Vehicle Standards and Highway Diesel Fuel Sulfur Control Requirements. To study non-neoplastic respiratory effects of DEP, a cross sectional study compared 137 DE exposed workers to 127 non-DE exposed workers, and compared lung function, cytokinesis-block micronucleus cytome index, and levels of urinary PAH metabolites. Pulmonary function tests results were all reduced in the DE exposure group, including decreased forced expiratory volume in 1 s and decreased ratio of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC), indicating obstructive lung changes. In addition, decreases in forced expiratory flow were associated with increased levels of PAH metabolites and cytokinesis-block micronucleus cytome index (p < 0.05) (Zhang et al., 2017). This study identifies the consequences of chronic DE exposure and how PAH levels can be used to indicate when exposure levels are significant enough to impart damage to the respiratory system.

3.3.1. Pathogenesis Epidemiological evidence suggests an association between benzene concentrations in ambient air and leukemia, but most of the mechanisms to explain the association has been conducted based on the association between occupational exposure to benzene and leukemia. Benzene is carcinogenic due to its ability to impart oxidative stress and DNA damage, but alterations in epigenetic regulation may also be involved. Philbrook et al. investigated if benzene exposure in utero contributed to DNA methylation or histone modifications in maternal bone marrow or fetal livers in mice. Hypomethylation was observed in maternal bone marrow (Jimenez-Garza et al., 2017; Philbrook and Winn, 2015), and while the exact mechanism is unknown, it may be related to increased reactive oxygen species that interfere with the transfer of methyl groups to DNA, leading to oxidative DNA damage (Weitzman et al., 1994). A study by Zhang et al. demonstrated that workers exposed to diesel engine exhibited significantly higher micronucleus, nucleoplasmic bridge, and nuclear budding frequencies in peripheral blood lymphocytes, suggesting diesel exhaust is associated with micronucleus changes (Zhang et al., 2015), thereby increasing the risk for hematopoietic cancers in DE exposed populations.

3.4.1. Pathogenesis Several major pathways have been implicated in the respiratory system in response to DE exposure. Alveolar macrophages were found to respond to in-vivo DE exposure through both ROS- and nitric oxidemediated pathways. DE exposure increases phagocytosis and leads to a time-dependent increase in the production of superoxide anions and mitochondrial dysfunction in alveolar macrophages. In addition, nitric oxide plays a pro-inflammatory role in the DE-exposed lung, including enhanced neutrophil recruitment, increased air/blood barrier leakage, and increased production of IL-12 in alveolar macrophages. The induction of the nitric oxide pathway helps to curb the effects of DEPinduced ROS production and its ability to lessen the removal of intracellular pathogens (Zhao et al., 2009). Several molecules are involved in the events responsible for DEinduced inflammation and injury. An in-vitro study investigated the effects of DE on intracellular adhesion molecule- 1 (ICAM-1) expression. DEP upregulated ICAM-1 mRNA levels and surface protein expression in human bronchial epithelial cells. ICAM-1 is thought to play an important role in the accumulation of inflammatory cells, and the present study found increased neutrophil attachment onto the DEP treated epithelial cells (Takizawa et al., 2000). Gowdy et al. investigated how exposure time and DEP levels affected inflammation and cytokine expression using an animal murine model. Neutrophil counts were increased immediately after a single exposure to 2.0 mg/ m3 of diesel emissions and continued to show a dose-dependent increase after 5 days and 18 h of exposure. Lung injury started immediately after 1 day of exposure and persisted through 5 days, with worse lesions in the 2.0 mg/m3 exposure group. TNF-alpha and IFNgamma were upregulated after five days of exposure to either 2.0 mg/ m3 or 0.5 mg/m3 of diesel emission particulates. In addition, surfactant proteins and mRNA expression were decreased after 5 days and 18 h of DE exposure at the lower concentration. The increase in inflammatory responses and the decrease in host defenses increases the susceptibility for respiratory pathogens and infection (Gowdy et al., 2008).

3.4. The respiratory system When encountering DE one of the first lines of defense is the respiratory system. As a major component of PM2.5, DE has been implicated in lung cancer in addition to non-neoplastic lung disease. A recent study used data and subjects from the Adventist Health Study-2 (Butler et al., 2008) to determine the association between PM2.5 and incident adenocarcinoma of the lung. They used a non-smoking population in which 81% had never smoked and 55% of former smokers had quite > 20 years ago. They found a 31% increase in incident lung adenocarcinoma associated with each 10 μg/m3 increase in ambient PM2.5 concentration (Gharibvand et al., 2017) (Fig. 2). The results from 256

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Fig. 2. PM and lung cancer. This figure demonstrates the incidence of lung adenocarcinoma cases associated with each 10 μg/m3 increase in ambient. PM2.5 concentration. Permission by Dr. Lida Gharibvand.

has also been implicated in inducing cytotoxic effects in bronchial epithelial cells (Totlandsdal et al., 2015), and can increase tracheal pressure via activation of the environmental sensor transient receptor potential 1 (TRPA1) (Robinson et al., 2017). In addition, TRPA1 activation has been shown to elicit a cough reflex (Birrell et al., 2009) (Fig. 3). Understanding how DE exposure is implicated in chronic pulmonary disease was recently investigated. An animal murine model studied whether the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is activated in an elastase-induced emphysema mice model after DEP exposure. NLRP3inflammasome expression and IL-1Βeta were increased after DEP exposure and were inhibited by the antioxidant N-acetylcysteine. To have full activation of the NLRP-3 inflammasome, upregulation of its components, including proIL1-β and proIL-18, needs to occur, followed by assembly into the inflammasome structure and release of proinflammatory interleukins. Oxidative stress is thought to be the trigger leading to inflammasome activation in the present study (Uh et al., 2017). The results provide a link between inflammasome activation in the presence of DEP exposure in an emphysema animal model and identified the benefits of antioxidants. Dietary intake of anti-oxidants in the presence of respiratory disease and DE exposure may provide significant benefits in reducing the inflammatory response and COPD exacerbation and is an area for future studies.

When studies started identifying DE as a pulmonary carcinogen (Mauderly et al., 1986; Mauderly et al., 1987; Stober, 1986) researchers began investigating the pathogenicity of lung cancer from DE exposure. An animal study using rats found that higher levels of total DNA adducts and exhaust-induced adducts were present in tissues where exhaust-induced tumors were identified (Bond et al., 1988). Direct intrapulmonary administration of 1,6-Dinitropyrene, a component of DE, resulted in a 1.8-fold increase in DNA binding at treatment doses up to 30 micrograms and mutations in T-lymphocytes increased with doses up to 100 micrograms (Beland et al., 1994). A study wanted to determine the role of oxygen radicals in lung carcinogenesis induced by DE. A dose dependent increase in of 8-hydroxy-deoxyguanosine (8OHdG) formation occurred following intratracheal injections of DEP. Oxygen radicals released during phagocytosis and generated by enzymatic and non-enzymatic reaction were thought to be related to the formation of 8-OHdG, a marker of oxidative DNA damage. Formation of 8-OHdG correlated with an increased tumor incidence, suggesting DNA damage is an important factor in lung carcinogenesis following DE exposure (Ichinose et al., 1997). Early pioneers in the study of DE identified the basic processes underlying the pathogenicity of DE as a carcinogen and fueled the movement for more strict emission standards and the designation of air pollution as a Group 1 carcinogen (IARC, 2013). More recent studies have investigated the specific components of DE and their effects on the respiratory system. Meldrum et al. used an animal murine model to investigate the effects of a dichloromethane extract of DE. The extract was characterized by the presence of PAHs and nitro-substituted PAHs. These components of DE inhibit CXCL10, a chemokine that regulates the recruitment of type 1 CD4 + T helper cells to the site of injury, indicating the capacity of DEP to modulate the immune system responses in bronchial epithelium. DEP exposure may limit Th1 expression, allowing overexpression of the Th2 airway inflammation in allergic airway diseases to (Meldrum et al., 2017). PAH

3.5. Cardiovascular system Prospective data from a cohort of women was used in a study to investigate the associations between air pollution and all-cause and cause-specific mortality, particularly cardiovascular, pulmonary, or cardiopulmonary causes, or lung cancer. Individuals in sub cohorts were followed for 18 years in North Rhine-Westphalia in Germany and residential mobility, vital status, and address information were 257

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Fig. 3. DE exposure and induction of sensory fibers in the production of cough. This figure demonstrates the process by which DE induces the afferent fibers/C-fibers to stimulated cough and bronchospasm via ROS and TRPA1. DE with PAH evokes activation of airway C-fibers. PAHs are major constituents of DEPs and activate aryl hydrocarbon receptor (AhR) and subsequent mitochondrial reactive oxygen species (ROS) production. ROS activate transient receptor potential ankyrin-1 (TRPA-1) on nociceptive C-fibers, leading to respiratory symptoms.

index, end-diastolic and end systolic diameter were of greater magnitude than when comparing highways with limited access and roads together. These results suggest an association with systolic dysfunction and cardiac remodeling in individuals living closer to major roadways (Weaver et al., 2017). A cross-over study used healthy non-smoking adults to examine the impact of physical activity on the relationship between traffic related air pollution and heart rate variability in Barcelona, Spain. They were exposed to 4 levels of air pollution at 4 different sites. Overall, inverse associations between air pollution and heart rate variability were stronger at lower exposure concentrations and then shifted to a more positive association at higher concentrations. Concentration of UFPM and PM2.5 at low traffic sites were associated with reductions in heart rate variability during physical activity, while physical activity at the high traffic sites reduced the impact of air pollution on heart rate variability, as no associations were observed. These findings suggest that physical activity may offset the detrimental impact of air pollution on parasympathetic modulation of the heart at higher exposure levels. Additionally, physical activity may decrease the impact of air pollution on heart rate through distraction (Cole-Hunter et al., 2016). Another factor to consider is different dose potentials of specific components of traffic related air pollution, including UFPM and black carbon, as UFPM can more easily penetrate the cardiovascular and central nervous system compared to PM2.5 and black carbon (Knibbs et al., 2011). Another study used healthy non-smoking women in Montreal, Canada to investigate whether traffic related air pollution contributed to significant cardiovascular effects during physical activity. The study used micro-vascular function, blood pressure, and heart rate variability as indicators to determine whether pathophysiological cardiovascular mechanisms are linked to air pollution. Average concentrations of UFPM and PM2.5 were measured during each 2-hour exercise period. This study found decreased reactive hyperemia index values 3 h after

obtained for nearly 5000 women. Yearly average concentrations of NO2 and PM10 were used to estimate the women's exposure. The women included in the study were living in industrial and non-industrial cities with varying concentrations of NO2. As supported by numerous cohort studies (Abbey et al., 1999; Dockery et al., 1993; Filleul et al., 2005; Hoek et al., 2002; Miller et al., 2007; Pope 3rd et al., 2002), PM10 exposure has a statistically significant effect on cardiopulmonary mortality and all-cause mortality. The study found that long-term NO2 exposure was associated with increased all-cause mortality, as was living closer to major roads (> 5000 vehicles/day). The present study's results did not account for socioeconomic status or lifestyle habits (Heinrich et al., 2013). In this study, baseline data collection went from 1985 to 1994, and individuals were followed until 2008. Additionally, the women were 50–59 years old at the time data collection started and had therefore likely been exposed to air pollution starting in the 1930s. It is important to consider the air quality during that time, as air pollutants from vehicles have only been regulated in the European Union since the 1970s (German Partnership for Sustainable Mobility, 2014). In order to establish an association between heart failure and air pollution, investigators used data from the Jackson Heart Study to conduct a cross sectional study. The data included a cohort of AfricanAmerican men and women living in a metropolitan area in Mississippi. Residential distances to the nearest major roadway was used as a marker of long-term exposure to traffic-relation pollution. Indicators of cardiac structure included left ventricular hypertrophy, end-diastolic diameter, and end-systolic diameter, as measured by ECG. After excluding individuals with previously diagnosed cardiac disease, the majority of participants living > 1000 m from a major roadway, with only < 2.1% living < 150 m away. Those living < 150 m from a major roadway had left ventricles that averaged a 1.2 mm greater end-systolic diameter. When isolating the residential distance from a primary highway with limited access, associations with left ventricular mass 258

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mediated through activation of the aryl hydrocarbon receptor, whose signaling is important in extracellular matrix remodeling. Indeed, compared to the control group, aryl hydrocarbon receptor expression was increased by 19% after exposure to DE. This lead to subsequent decreased expression of hypoxia inducible factor 1α and one of its downstream mediators, VEGF. In addition, DE reduced cardiac interstitial collagen by 23%, leading to ventricular dilation, as evidenced by echocardiograph (Bradley et al., 2013). A recent study found the toxic effects of PAH were mediated by a nontransciptional signaling pathway for the aryl hydrocarbon receptor located in mitochondria (Robinson et al., 2017). This suggests PAH may be one of the components in DE contributing to the sequalae of cardiovascular dysfunction. Due to the complex nature of DE, combining studies examining organ-specific effects following DE exposure with studies identifying individual components of DE and their molecular effects will continue to guide ongoing attempts to improve the components of DE and reduce the risk for disease.

exposure to UFPM, indicating an effect on vasomotor function. Neither pollutant demonstrated significant changes in blood pressure. In addition, decreased heart rate variability occurred during PM2.5 exposures with physical activity, reflecting decreased parasympathetic modulation (Weichenthal et al., 2014). It is important to study and understand the effects of DE under various conditions, as certain individuals may be even more susceptible to these effects, including those with pre-existing cardiovascular or pulmonary disease. There may be even less modulatory responses in diseased states, making these individuals more susceptible to the biological effects of PM. 3.5.1. Pathogenesis To establish cardiotoxicity related to diesel exhaust exposure, an animal study measured the reserve of refractoriness, or the stability of the heart's electrical signals through analyzing the underling heart tissue actions potentials. After subjecting rats to a single air pollution exposure of 150 micrograms of PM2.5/m3, DE exposure caused significant increases in the risk of cardiac conduction instability in both normal and hypertensive rats. The effects in rats during DE exposure were not evident until 24 h later when compared to controls, suggesting both latent and subtle electrophysiological changes that can be triggered later. Though the results after 24 h were of subclinical nature, it generates an arrhythmia substrate that, if combined with a stressful event, exercise, or an immune activation, could initiate a clinical event (Hazari et al., 2017). Robertson et al. identified pulmonary vanilloid receptor TRPVI, the sympathetic nervous system, and local oxidative stress as mediators of myocardial injury following DE exposure. Moreover, elevated systolic blood pressure was improved following TRPV1 receptor blockage. Following ischemic injury with reperfusion, ischemia-associated arrhythmias were prolonged with increased severity in rats exposed to DE, frequently leading to death. This outcome was also suppressed by pulmonary administration of a TRPV1 antagonist, suggesting sensory nerve activation in mediating the effect of DE (Robertson et al., 2014). An animal study used an endothelial tube model to better understand how cytokines modulate endothelial permeability after DE exposure. DE exposure induced intracellular reactive oxygen species in the tube cell in a dose dependent manner. This was followed by the release of pro-inflammatory TNF-α and IL-6. Both cytokines upregulate vascular endothelial growth factor A (VEGF-A) which induces vascular permeability. Interestingly, administering N-acetyl cysteine into the tube cell completely blocked oxidative stress and showed reduced levels of oxidized glutathione (GSSG) levels compared to the DE group, and increased amount of reduced glutathione (GSH), thereby offering a level of protection against the DE-induced oxidative stress (Tseng et al., 2015). Studies that incorporate different methods to ameliorate the damaging effects of DE provide additional clues into the detailed mechanisms related to DE exposure. Particulate matter from DE not only directly affects the heart, but also indirectly via lung-mediated effects. An animal study performed in vitro experiments using rat cardiomyocytes and lung epithelial cells. Direct treatment of cardiomyocytes with DE caused contractility dysfunction, but cardiomyocytes were also affected after lung epithelial cells were treated with DE. Monocyte chemoattractant protein-1 (MCP1) is released from the lung epithelial cells in response to PM exposure and activated the MCP-1/CCR2 receptor, leading to cardiovascular dysfunction and increased serum cytokines. Like Tseng et al., investigators were able to decrease contractile dysfunction of DE by treating the cardiomyocytes with antioxidants before culturing with DE (Gorr et al., 2015). This study demonstrated how DE exposure leads to oxidative stress, but also the downstream inflammatory effects and cellular damage, as evidenced by cardiomyocyte contractile dysfunction. However, these studies were not able to characterize which components of diesel exhaust contributed to the dysfunction. An animal study investigating chronic exposure to DE hypothesized that cardiovascular dysfunction, including ventricular remodeling, is

3.6. The renal system Another organ system affected by DE exposure is the renal system. Though not often considered in large epidemiological studies, the kidney system is also at risk. A recent meta- analysis used data from 14 different European cohort studies to determine whether an association exists between outdoor PM and kidney parenchyma cancer. Though not statistically significant, hazard ratios were elevated for kidney parenchyma cancer incidence in association with higher concentrations of various measures of PM air pollution (Raaschou-Nielsen et al., 2017). Ongoing studies evaluating kidney function biomarkers following DE exposure is an area of importance, as human studies evaluating the effects of DE exposure are limited.

3.6.1. Pathogenesis Animal studies have shown how pre-existing renal disease increases the adverse effects of PM. This is especially the case with PM2.5, including DEP. A recent study found that a single exposure to DEP is a risk factor in subjects with acute renal failure using a rat model. They also found that thymoquinone, the active ingredient in Nigella Sativa seed oil, is a useful agent because of its anti-inflammatory actions. Thymoquinone was able to ameliorate the rise of enzymes indicative of tissue damage after exposure to DE (Ali et al., 2015), as it has already been implicated as a risk factor in healthy, hypertensive, and diabetic animal subjects (Nemmar et al., 2013). A more recent study in 2017 looked further into vascular damage associated with exposure to DEP in subjects with adenine-induced chronic kidney disease. By monitoring renal blood flow and blood pressure, they found that phenylephrineinduced decreases in renal blood flow and increases in blood pressure were significantly potentiated in subjects exposed to DEP. These symptoms were even worse in subjects with chronic kidney disease (Al Suleimani et al., 2017).

4. Biomarker evidence Studies that used biological markers provide a deeper level of knowledge into the pathogenesis of disease. They provide explanations to support epidemiological studies that continue to find associations between disease and air pollution. Studies conducted in the last 5 years using biological markers reinforce the epidemiological evidence that DE has on physiological systems. Using biomarkers is also a way to scientifically identify whether an individual has been exposed. Though the studies measured different indices and utilized a variety of biomarkers to show biological plausibility, they combine to impart a common theme that includes the induction of inflammation, oxidative stress, or cellular damage with DE exposure (Table 3). 259

Cardiovascular effects

Cardiovascular health

Adults with heart failure

Healthy adults

Adults Adults

Adults

Randomized, controlled, blinded crossover study NCT01960920

Randomized single-blind crossover intervention study NCT01570062

Randomized double-blind cross-over study

Randomized crossover-controlled exposure study

Randomized double-blinded controlled crossover study

260 Inflammation

Cardiovascular health Mitochondrial oxidative DNA damage indices

Children

Adults Mother and newborn

Adults Elderly Adults Healthy college-aged students Adults with chronic exposure vs. nonexposure

Prospective cohort study using subjects from the Barn/Child, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) cohort Cohort study (Framingham Heart Study) Cohort study

Cohort study

Cohort study

Prospective cohort (CoLaus) study

Healthy Volunteer Natural Relocation (HVNR) Prospective cohort study

Cross-sectional molecular epidemiology study

Inflammation and lung cancer risk

Systemic/hematologic effects Systemic/hematologic effects Cardiovascular effects

Urinary metabolites

Inflammation/neurotoxicity

Adults

Controlled, blinded crossover study

Pulmonary inflammation

Hematological/endothelial changes Allergic respiratory disease

Blood pressure and lung function

Adults

Randomized controlled trial NCT01874834

Indices measured

Subjects

Study model/population

Table 3 Studies investigating the organ-specific and systemic effects of DE through the use of biomarkers.

PM2.5 chemical constituents (metals, transition metals, and NO3−) DE (PM2.5)

PM10

PM2.5, CO, NOx, O3

Nitro-PAH in PM2.5

PM2.5 PM10 and PM2.5

NO2 and PM10 from road traffic

DE (PM2.5)

Allergen and PM2.5

Allergen and PM2.5

Diesel exhaust (PM2.5)

Traffic or wood smokeimpacted areas (PM2.5)

PM2.5 vs filtered air (FA)

DE and O3

Exposure

- ↑ IL-1β, IL-6, and TNF-α, cytokines secreted by macrophages and implicated in inflammatory response - ↑ PM2.5 associated with ↑ in circulatory biomarkers: TNF-α, Fibrinogen, PAI-1, t-PA - ↑ PM2.5 associated with ↓ in vWF and soluble platelet selectin - ↑ C-reactive protein - ↑ CCL15/MIP-1D Both markers are associated with the risk of lung cancer (28981818) - ↑ CXCL11/I-TAC - ↑ IL-16 Both function as attractants for WBCs

- ↓ reactive hyperemic index with ↑ PM2.5, NOx, CO exposure

(Stiegel et al., 2017)

- DE exposure altered FEV-1 and IL-8 trended linearly, and IL-12 showed an inverse trend - O3 exposure reduced FEV-1 along with IFN-gamma and TNF-α, - DE + O3 exposure reduced FEV-1 and increased expression of IL-2 and IL-8 - Systolic BP varied symmetrically about zero and Diastolic BP exhibited wide range of change - ↓ reactive hyperemic index and ↑ brain natriuretic peptide (BNP) by 41.5% after PM2.5 inhalation - ↓ BNP by 33.8% with FA - No changes in heart rate variability - No change in hematologic indices in peripheral blood - ↑ C-reactive protein in traffic exposure group. - No association with wood smoke exposure - No association with endothelial function, IL-6, or band cells in either exposure group - ↑ hematocrit; no significant increase in hemoglobin levels - No DE effect on WBC, neutrophils, lymphocytes or erythrocytes. - Prior exposure to allergen or DE significantly altered global DNA methylation in the human bronchial epithelium upon subsequent exposure 1 mo. later - DE does not augment allergen effect on serum adipokines - Co-exposure in normal airway responsiveness: high Adiponectin (anti-inflammatory)/Leptin (pro inflammatory) ratios - Co-exposure in hyper-responsive airways: low Adiponectin/Leptin ratios - No changes in IL-6, or TNF-α, indicators for inflammation. No changes in astrocytic protein S100B, neuronal cytoplasmic enzyme neuron-specific enolase, or serum brain-derived neurotrophic factor after acute exposure - ↑ IL-6, especially in asthmatic children (8 y/o) - ↑ IL-10 in asthmatic children (8 y/o) - ↑ IL-10, 13, and TNF-α in 16 y/o children exposed in infancy - No association with Coronary artery calcium score - ↑ 8-OHdG in maternal blood with exposure to PM10 in 3rd trimester and with exposure to PM2.5 the entire pregnancy - ↑ 8-OHdG in umbilical cord blood with exposure to PM10 in 1st and 2nd trimester - ↑ 8-hydroxy nitropyrene in urine

(continued on next page)

(Bassig et al., 2017)

(Wu et al., 2012)

(Tsai et al., 2012)

(Miller-Schulze et al., 2016) (Zhang et al., 2016)

(Dorans et al., 2016) (Grevendonk et al., 2016)

(Gruzieva et al., 2017)

(Cliff et al., 2016)

(Kramer et al., 2017)

(Clifford et al., 2017)

(Krishnan et al., 2013)

(Kajbafzadeh et al., 2015)

(Vieira et al., 2016)

Study

Physiologic/biomarker findings

H. Reis et al.

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Legal initiatives and measures to reduce pollutant emissions has contributed to improved air quality and more strict standards for diesel engines. The European Union developed standards and objectives for pollutants. Currently, NOx emissions from road transport contribute to 39% of the EU emissions, largely due to the growth in transport and the increased amount of diesel vehicles (EEA, 2016). Directive 2008/50/EC introduced PM2.5 objectives for each member state, targeting the exposure of the population to fine particles. In 2015, the aggregate European Union emissions of NH3 and PM2.5 were almost at the 2020 reduction commitment set for these pollutants. The National Emissions Ceilings (NEC) Directive came into effect in 2016 and set the 2020 and 2030 emissions reduction commitment for NOx, non-methane volatile organic compounds, SO2, NH3 and PM2.5, and carbon monoxide (CO). Along with the new standards, each NEC Directive Member States have to report their individual plans for air pollution control and how they will reach their 2020 and 2030 commitments (EEA, 2017). In China, the focus has been on implementing an early-warning evaluation system for heavy air-pollution weather using a consistent and standard grading system. In 2016, the “Five-Year Plan” on Eco-environmental protection allocated 11.2 billion Yuan for the prevention and control of air pollution. 75% of the cities at and above prefecture level (338 cities), failed to meet national ambient air quality standards. However, PM2.5 and PM10 were down approximately 6% compared to 2015 (Ministry of Environmental Protection, 2017). Since 2007, the EPA in the United States continues to adopt more stringent standards to dramatically reduce emissions of diesel PM and NOX from diesel engines. This included a new regulation to monitor emission control systems on large highway diesel and gasoline trucks for malfunctions, including a program designed specifically for marine diesel engines to reduce NOx and PM emissions. DE from post-2006 and older retrofit diesel engines contain technological advancements such as electronic controls, ultra-lowsulfur diesel fuel, oxidation catalysts, and wall-flow diesel particulate filters (NTDE). The continued efforts to reduce emissions were fueled by close to $50 million in funding under the Diesel Emissions Reduction Program, created under the Energy Policy Act of 2005 (Agency, 2017). Initiatives across the world are analyzing data on air pollution, including the components and the sources, and responding with tighter restrictions, closer monitoring, and advanced technologies to improve air quality. Part 1 of the Advanced Collaborative Emissions Study (ACES) aimed to characterize the emissions and health impact of NTDE using indicators of lung toxicity in animal models. Rats were exposed to 2007 engine exhaust for up to 28 months for males and 30 months for females. NO2 concentration level was the primary indicator of pollution in the present study. Exposure to NTDE did not increase risk for tumor formation (McDonald et al., 2015), whereas earlier DE was known to cause lung tumors. When comparing to previous studies that analyzed effects of traditional DE (Heinrich et al., 1986; Mauderly et al., 1994), use of NTDE resulted in less accumulation of diesel particulate matter in lung macrophages. The lesions noted on histopathology were not preneoplastic and occurred only at the highest exposure levels. The lesions were described as reactive in nature and with terminal bronchiole and alveolar duct epithelial proliferation uniformly occurring at the centriacinus, a region involved in gas exchange. Biomarkers for oxidative stress and inflammation were elevated, including IL-6, keratinocytederived chemokine, and total white blood cells, and macrophages. Macrophage accumulation occurred only at the highest exposure levels, and was found in 17% of the male rats, with a mean lesion severity of 1.1 out of 4. A mild yet progressive decline in pulmonary function was observed and manifested more strongly in females than males. Overall, it is thought that the advanced technologies reduce the amount of PM mass and emissions, and that NO2 is a major contributor to the biological responses, as results were similar to previous studies in which rats were chronically exposed to isolated NO2 (McDonald et al., 2015). Of

This table includes studies that investigated various biomarkers to indicate biological plausibility of the effects associated with exposure to diesel exhaust. Abbreviations 8-OHdG: Mitochondrial 8-hydroxy-2′-deoxyguanosine CCL15: Chemokine (C-C motif) ligand 15/Macrophage inflammatory protein (MIP) CXCL 11: C-X-C motif chemokine 11 (CXCL11) that is also called Interferon-inducible T-cell alpha chemoattractant (I-TAC) PAI-1: plasminogen activator inhibitor type 1 sICAM-1: soluble intercellular adhesion molecule

Adults Cross-sectional study

Systemic/hematologic effects

PM2.5

(Chiu et al., 2016)

- ↑ sICAM-1 with ↑ organic carbon, PM2.5 and elemental carbon ➔ present on endothelial cells and facilitates leukocyte adhesion and migration, a marker of inflammation - No association with C-reactive Protein or IL-6 - ↑ PM2.5 associated with ↑ in inflammatory markers Adults Cross-sectional study

Cardiovascular Effects

Elemental Carbon, Organic Carbon, PM2.5

Study Physiologic/biomarker findings Exposure Indices measured Subjects Study model/population

Table 3 (continued)

(Zhao et al., 2013)

5. NTDE and multi-system effects

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workplace and on the roads, including using special fuel additives such as biodiesel (OSHA, 2012). These recommendations warrant investigation into biodiesels and petroleum and their effects on organ systems compared to traditional DE. An animal study compared cardiovascular responses to emissions from different types of biodiesel and compared them to petroleum diesel. Both pure soy biodiesel and petroleum diesel increased heart rate variability, indicating changes in autonomic tone. In addition, pure biodiesel increased serum C-Reactive Protein with repeated exposure to 50 micrograms/m3 combustion emissions, and increased LDL cholesterol and decreased HDL cholesterol at 150 and 500 micrograms/m3. DE increased only triglycerides after repeated exposure to 50 micrograms/m3 of combustion emissions. Mixed biodiesel did not demonstrate statistically significant cardiotoxic effects (Farraj et al., 2015). Another study compared biodiesel emissions to traditional DE. Using the bronchoalveolar lavage fluid marker γ-glutamyl transferase and aorta markers, a study examined pulmonary, systemic, and cardiovascular effects after exposure to mixed (20% soy biofuel plus 80% low sulfur petroleum diesel), pure biodiesel exhaust, and traditional DE. In their results, petroleum DE induced greater pulmonary injury and neutrophilic inflammation as determined by increased GGT activity, an enzyme involved in amino acid transport for glutathione synthesis. As for the heart, pure biodiesel induced a vascular response immediately after a single 4-hour exposure in healthy rats, suggesting it could create a greater acute systemic vascular impact than petroleum DE. In addition, endothelin-1 expression was increased in the aortas of hypertensive rats exposed to pure biodiesel at the 4-week time point (Bass et al., 2015). Continued evidence- based studies on alternative fuels may provide global industry with future options to reduce PM, but basic science reports of potentially toxic cardiovascular and systemic effects of alternative fuels should not be overlooked. This includes determining the components of the biodiesels contributing to the toxic effects, determining if mixing biodiesel with low sulfur diesel consistently results in less pulmonary or cardiovascular effects, and if so, what makes the mixed fuel protective. To thoroughly investigate DE and alternative fuel emissions, developing technology to distinguish source contributions is an important facet in environmental science and research. Currently, new methods include laser, spectroscopic, and optoelectronic methods to identify sources and exposure levels of pollution. Though bio-based energy sources are an emerging idea to help control air pollution, continued study and research in this area will help decipher the risks and benefits associated with alternative fuel and energy sources to combat global air pollution (Weidemann et al., 2016).

note, between 2007 and 2010, engine technology was updated in response to a new NOx standard getting phased-in. By 2010, all engines were required to comply with the 0.2 g/bhp-hr NOx limit, resulting in further reductions in NOx emissions (EPA, 2017). Part 4 of the ACES bioassay analyzed NTDE and cardiovascular health. Rats were exposed for 24 months to filtered air or DE, and investigators examined if plasma markers changed in an exposure level-, sex-, or exposure-duration-dependent manner. These markers included vascular inflammation, thrombosis, and cardiovascular aging. There were no changes in prothrombotic markers or cardiac effects after 2 months of NTDE exposure. After 12 months of exposure, no significant changes were noted. However, at 24 months, female rates were observed to have increased IL-6 and plasma sICAM-1 after 24 months of mid-and high-level DE exposure. Elevation of these two markers suggest vascular inflammation and endothelial injury, however the current study was not able to determine the specific cell tissue or organ source generating expression of Il-6 of sICAM-1. Cholesterol and non-HDL cholesterol were decreased in female rats exposed to high-level DE for 24 months, and serum triglycerides were decreased in female rats exposed to mid-level DE for 24 months. Overall, there was high variability with respect to plasma triglyceride levels and DE exposure at 24 months, and this may have been related to the non-fasting state of rats prior to euthanasia and blood collection. Overall, NTDE exhibited limited effects on the aging cardiovascular system after 2 years of exposure in the current study, but investigators did not test the effects of chronic NTDE exposure using a susceptible-animal model in which the subjects may have had increased susceptibility to chronic exposure (Conklin et al., 2015). Overall, it is thought that the advanced technologies reduce the amount of PM mass and emissions. Even more, other components of DE known to be carcinogenic or had toxic properties were also reduced relative to the pre-2007 diesel engines (Costantini et al., 2016). The ACES studies on the respiratory and cardiovascular effects of NTDE provide a new framework for future studies to operate, and that is with the use of NTDE from low sulfur fuel containing less NOx emissions. Hallberg et al. examined the risk of exposure to NTDE in rodent model to assess overall genotoxicity. Genotoxicity was defined as DNA strand breaks in lung tissue. Concentrations of 8-OHdG were measured as well to determine the presence of oxidative damage. After 1 and 3 months of exposure, small increases in 8-OHdG were noted. Overall, the study found no significant increases in oxidative damage or DNA strand breaks at any pre-determined end point of the study, suggesting an improvement in genotoxicity effects of NTDE (Hallberg et al., 2015). Another outcome measure in the ACES study was micronucleus formation after chronic exposure to NTDE. Exposure to NTDE for up to 2 years was not associated with genotoxic changes, measured by the amount of micronuclei-containing reticulocytes (Bemis et al., 2015). Discussion needs to focus on the continued evaluation of NTDE and its influence on long-term health and air quality, and the importance of ongoing efforts to reduce pollution and carbon emissions from diesel exhaust. (McClellan et al., 2012).

6.2. Additional factors The use of anti-inflammatories has emerged as an area of concentration in the battle against the harmful effects of DE and PM. An animal study investigated the use of emodin on the acute (24 h) effects of DE. Emodin is commonly found in the roots of the rhubarb plant and has strong anti-inflammatory and antioxidant effects. When tested in rats after pulmonary exposure to DE, emodin prevented an increase in cardiac proinflammatory cytokines, including TNF-α and IL1-β. Regarding the prothrombotic complications of DE, mice treated with emodin in vitro prior to DE exposure had a statistically significant reduction in platelet aggregation. The emodin prevented a decrease in activated partial thromboplastin time and prothrombin time (Nemmar et al., 2015). Unfortunately, current studies exploring the benefits of antioxidants in combating the negative effects of DE have come up short. A recent double-blinded crossover human study measured changes in brachial artery vasoconstriction following DE exposure and found no changes with pretreatment of an antioxidant. In fact, pretreatment with vitamin C and N-acetyl cysteine resulted in greater DEinduced vasoconstriction. This randomized controlled trial stratified subjects based on genotype. Those with polymorphisms of the TRPV1 or

6. Diesel exhaust exposure and future directions 6.1. Alternative fuels Preventing and minimizing the exposure is the key in reducing or eliminating DE related negative health consequences. Efforts have been made to reduce exposure to bus diesel exhaust in school aged children. A school district in North Carolina, U.S.A. has worked to improve air quality and safety through nurse education, implementation of environmental health strategies, and legislative initiatives including trading in diesel busses for compressed natural gas busses (Mazer et al., 2014). Additionally, biodiesel fuels have emerged as an alternative to the PM-emitting petroleum-based diesel fuels. The Occupational Safety and Health Administration (OSHA) promotes effective strategies for the 262

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AGTR1 gene showed greater vasoconstriction after DE exposure, as polymorphisms correlate with an elevated blood pressure (Sack et al., 2016). It is important to continue designing study trials evaluating the efficacy and benefits of anti-inflammatories as a preventive measure for DE exposure. Understanding how genotypes alter an individual's susceptibility to the harmful effects of DE exposure will improve prevention and facilitate individualized treatment plans.

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