ANTIOXIDANTS & REDOX SIGNALING Volume 21, Number 1, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2013.5694
NEWS & VIEWS
Does Enhancement of Oxidative Stress Markers Mediate Health Effects of Ambient Air Particles? Pier Alberto Bertazzi,1 Laura Cantone,1 Pasquale Pignatelli,2 Laura Angelici,1 Valentina Bollati,1 Matteo Bonzini,3 Michele Carugno,1 Pier Mannuccio Mannucci,4 and Francesco Violi 2
Abstract
Evidence indicates that oxidative stress generation may contribute to health effects associated with particulate matter (PM) exposure. We investigated oxidative stress markers in 113 workers exposed to metalrich PM and 61 nonexposed comparable volunteers. The plasma levels of soluble NOX2-derived peptide (sNOX2-dp) and two oxidative stress markers, urinary 8-hydroxydeoxyguanosine (8-OHdG) and 8iso-prostaglandinF2alpha (8-iso-PGF2a), were analyzed. The plasma levels of the antioxidant alphatocopherol were also evaluated. The workers’ average exposure to PM with an aerodynamic diameter < 10 lm (PM10) was much higher at the workplace than in the city, where the volunteers lived. Workers had significantly higher urinary 8-iso-PGF2a and 8-OHdG and plasma sNOX2-dp levels than nonexposed subjects. Alpha-tocopherol was much lower in workers compared with nonexposed subjects. In multivariable regression models adjusted for age, body mass index, and smoking, 8-iso-PGF2a increased in workers in association with PM10 and metal exposure; 8-OHdG and sNOX2-dp were associated only with iron. Alpha-tocopherol was inversely associated with each of the oxidative stress markers. Our observation leads to the hypothesis that the enhancement of oxidative stress markers associated with exposure to high metalrich PM levels represents a possible step in the pathways leading from particle exposure to systemic (e.g., cardiovascular) effects. Antioxid. Redox Signal. 21, 46–51.
Introduction
Innovation
E
pidemiological studies have repeatedly linked environmental exposure to particulate matter (PM) with increased mortality and morbidity from respiratory and cardiovascular disease, in particular, myocardial infarction and ischemic stroke (2). PM metal components have been shown to play key roles in determining PM-related cardiovascular effects. The mechanisms linking the inhalation of air particles to cardiovascular diseases are not completely understood, but PM-induced systemic inflammation has been suggested as a primary mediator. In addition, local and systemic oxidative stress has emerged as a likely common link between pulmonary exposure and systemic effects, including those on the cardiovascular system (2).
Extensive evidence indicates that the generation of oxidative stress contributes to the health effects associated with particulate matter (PM) exposure. We provide evidence that in subjects exposed to high levels of metal-rich PM, the soluble NOX2-derived peptide (sNOX2-dp), urinary 8-hydroxydeoxyguanosine (8-OHdG), and 8-iso-prostaglandinF2alpha (8-isoPGF2a) levels were elevated after controlling for age, smoke, and body mass index, suggesting a role for this enzymatic pathway in reactive oxygen species (ROS) overproduction. In addition, our findings suggest that following the PM stimulus for ROS formation, alpha-tocopherol is lowered as a consequence of its consumption.
1
Department of Clinical Sciences and Community Health, Center of Molecular and Genetic Epidemiology, Universita` di Milano and Fondazione IRCCS Ca` Granda Ospedale Maggiore Policlinico, Milano, Italy. 2 Clinica Medica, Sapienza, University of Rome, Roma, Italy. 3 Department of Clinical and Experimental Medicine, University of Insubria, Varese, Italy. 4 Scientific Direction, Fondazione IRCCS Ca` Granda, Ospedale Maggiore Policlinico, Milan, Italy.
46
OXIDATIVE STRESS, PM, AND CARDIOVASCULAR RISK
PM has been shown to produce oxidative stress in both in vitro and in vivo systems. In vitro studies have demonstrated a clear link between PM actions and levels of reactive oxygen species (ROS), and in vivo studies have consistently found evidence for increased levels of oxidative stress in PM-exposed animals. In rodents, a time- and dose-dependent production of ROS (i.e., superoxide and H2O2) was observed after exposure to concentrated ambient air pollution particles. The components in inhalable PM associated with ROS generation include metals (4). Indoor work conditions may cause a considerably higher exposure to PM than outdoor conditions. For example, in steel plants, workers are exposed to inhalable PM at levels well above the concentrations found in outdoor ambient air, and the PM mixtures contain a large proportion of potentially toxic metal components. In addition, in epidemiological studies, steel workers have been found to be at a higher risk for cardiovascular disease (1). Based on these studies, we selected a steel production plant with a wide range of exposures to well-characterized PM concentrations as a model to investigate whether PM and PM metal components induce changes in the following three oxidative stress markers: the plasma catalytic subunit of NADPH oxidase (NOX2), urinary 8-hydroxydeoxyguanosine (8-OHdG), and urinary isoprostanes (8-iso-PGF2a). Among the various enzymes that generate (intra)cellular ROS, the phagocyte NOX2 has the capacity to transport electrons across the plasma membrane and to generate superoxide and other downstream ROS. An increased NOX2 activity contributes to many health consequences, in particular, cardiovascular disease (7). ROS, produced either endogenously or exogenously, can simultaneously attack lipids, proteins, and nucleic acids in living cells. 8-OHdG, an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. The urinary excretion of 8-OHdG has been often used as a biomarker to assess the extent of repair of ROS-induced DNA damage in both clinical and occupational settings (5). Isoprostanes are prostaglandin-like substances produced in vivo independently of cyclooxygenase enzymes, primarily by free radicalinduced peroxidation of arachidonic acid (8). In particular, F2-isoprostanes are considered the best available biomarkers of oxidative stress status and lipid peroxidation in vivo because other products of the isoprostane pathway, such as D2- and E2-isoprostanes, are less suitable as they are less stable (8). Altogether, substantial evidence has indicated urinary 8-OHdG and isoprostanes as biomarkers of generalized cellular oxidative stress (5). Participants’ characteristics and exposure levels
The mean age of the 113 enrolled workers was comparable to that of nonexposed subjects. The proportion of current smokers and the average body mass index (BMI) did not differ between the two groups. The participants’ characteristics are reported in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/ars). The study workers displayed wide ranges of PM exposure. The highest mean levels of PM-associated metals were measured for zinc, iron, and lead. Details are provided in Supplementary Table S2.
47 Oxidative stress markers and alpha-tocopherol
In exposed workers, urinary 8-OHdG strongly correlated with urinary 8-iso-prostaglandinF2alpha (8-iso-PGF2a) and plasma soluble NOX2-derived peptide (sNOX2-dp). The correlation between urinary 8-iso-PGF2a and plasma sNOX2-dp was also statistically significant. In nonexposed volunteers, these stress oxidative markers were not significantly correlated. Workers had significantly higher urinary 8-iso-PGF2a, urinary 8-OHdG, and plasma sNOX2-dp than nonexposed volunteers. The differences between the oxidative stress markers in the two groups were statistically significant. The plasma levels of alpha-tocopherol were considerably lower in exposed workers compared with nonexposed volunteers. Raw data and statistics are provided in Supplementary Table S3. Association of oxidative stress markers with total PM mass and exposure to metal components
In exposed workers, urinary isoprostanes increased in unadjusted models in association with PM1 and several metal components. The model adjusted for age, BMI, and current smoking status confirmed this observation (Fig. 1). In particular, 8-iso-PGF2a increased in association with PM10, cadmium, cobalt, iron, lead, tin, barium, nickel, copper, and antimony (see Supplementary Data). When the association between urinary 8-OHdG and exposure was examined, an association was observed with iron. Plasma sNOX2-dp was associated with iron and copper (see Supplementary Data). Association of oxidative stress markers with plasma alpha-tocopherol levels
In exposed workers, multivariable regression models indicated that alpha-tocopherol had a significant negative association with urinary 8-OHdG, plasma sNOX2-dp, and urinary 8-iso-PGF2a (Fig. 2). In unexposed subjects, only urinary 8iso-PGF2a displayed a statistically significant negative association with alpha-tocopherol (see Supplementary Data). Discussion
The induction of oxidative stress and inflammation are important mechanisms that mediate the health effects caused by inhaled particles (6). In this study, we examined markers of oxidative stress, such as urinary isoprostanes and 8-OHdG, and found that subjects exposed to high levels of metal-rich PM in an occupational setting demonstrated enhanced oxidative stress compared with subjects living in a metropolitan area with no occupational exposure. This observation indicates that metalrich PM may be a stimulus for enzymatic pathways that ultimately result in ROS formation. To further explore this hypothesis, the circulating levels of NOX2, one of the most relevant cellular producers of ROS, were measured. In this study, we provide the first evidence that the NOX2 activity is increased in subjects exposed to metal-rich air polluting PM, suggesting a role for this enzymatic pathway in ROS overproduction. Several cellular lines may contribute to NOX2 upregulation. The assay used in this study mainly reflects NOX2 activation in blood cells, such as platelets, leucocytes, and monocytes, and it is conceivable that more than one cellular line contribute to NOX2 upregulation and, in turn, to ROS overproduction.
48
BERTAZZI ET AL.
FIG. 1. Multivariable linear regression models adjusted for age, body mass index (BMI), current smoking status (Yes/No). *p-Values £ 0.05. Geometric mean ratio (GMR) and 95% confidence interval (CI) expressing the changes in oxidative stress markers associated with an increase in exposure equal to the difference between the 90th and 10th percentile of the exposure distribution.
Consistent with this idea was the observed correlation in exposed workers for both urinary markers 8-OHdG and isoprostanes with NOX2 plasma levels. The direct association between serum NOX2 and urinary isoprostane is in concordance with previous reports showing that downregulation and upregulation of NOX2 were associated with decreased and enhanced formation of isoprostane, respectively, identifying NOX2 as one of the most important contributors to isoprostane formation (7).
We investigated whether the type and composition of PM affected oxidative stress. Urinary isoprostanes (8-iso-PGF2a) were associated with the finest particles (PM1) and several metal components, in particular, iron. Urinary 8-OHdG and plasma sNOX2-dp were also associated with iron. Iron is essential for metabolic processes and normal cellular functioning. However, increased availability of iron may modulate the production of ROS via the Fenton reaction and induce
FIG. 2. Multivariable linear regression models adjusted for age, BMI, and current smoking status (Yes/No). GMR [ = exp(b)] and 95% CIs. (GMR-1)*100 expresses the percentage change in oxidative stress markers associated with one lmol/mmol chol increase in alpha-tocopherol.
OXIDATIVE STRESS, PM, AND CARDIOVASCULAR RISK
oxidative injury and cellular toxicity. The increase of oxidative stress markers observed in this study may be associated with bioavailable iron from inhaled particle mixtures, suggesting an overload of inhaled iron as a critical toxicant for normal biological processes. New and relevant information stems from the present analysis of plasma levels of alpha-tocopherol, an antioxidant inhibiting lipid peroxidation (9). Alpha-tocopherol/ plasma cholesterol levels, the most reliable method for measuring alpha-tocopherol in blood, were greatly reduced in workers exposed to metal-rich PM and inversely associated with markers of oxidative stress. This observation suggests that upon continuous exposure to air pollution and ensuing stimulation for ROS formation, alpha-tocopherol is lowered as a consequence of its consumption. An intriguing consequence of this argument is the possibility of blunting ROS overproduction by air pollution and its potentially deleterious effect on the human body by alpha-tocopherol supplementation. This study has inherent limitations. Although workers were operating in a modern steel plant with state-of-the-art systems for exposure control, we cannot exclude that exposure to factors other than PM and metals, such as heat, carbon monoxide, and nonionizing radiation, might have contributed to the observed effects (3). Only area and nonpersonal measurements were available for exposure assessment. A further limitation was that unexposed subjects represented a convenience sample because of the known difficulty in obtaining a nonexposed reference sample when conducting studies on working populations. However, the characteristics of the two male samples in terms of age, BMI, tobacco smoking, and health conditions were comparable. Hence, these limitations are unlikely to seriously detract from the validity of our findings. The investigation of mechanisms linking the inhalation of air particles to cardiovascular disease is considered a high priority of biomedical research (2). The exploration of mechanisms involving alterations in oxidative stress markers and alpha-tocopherol in this study may help shed new light on the chain of events that unfold from particle exposure resulting in cardiovascular disease. Our findings could lead to the identification of potentially reversible alterations as potential targets for secondary prevention interventions, including supplementation with alpha-tocopherol. Notes Study population
We recruited 113 healthy active males aged 20 to 60 years, apparently free from disease, who worked in a steel production plant in Italy for at least 1 year. Individual written informed consent and approval from the local Institutional Review Board were obtained before the study. A selfadministered questionnaire was used to collect detailed information on smoking habits, drug use (including nonsteroidal anti-inflammatory drugs (NSAID), underlying medical conditions, BMI, and educational levels. Records from the factory administrative files were used to extract information on occupational history. Biological specimens were collected for each subject, including blood in EDTA tubes and urine morning spot samples. A whole blood aliquot (300 ll) was used for a complete blood count to obtain the absolute number and percentage of
49
granulocytes. The remaining whole blood was centrifuged on site (in an appropriately protected and equipped room in the plant infirmary, within 30 min of blood drawing) at 2500 rpm for 15 min to separate platelet-poor plasma. A plasma aliquot (200 ll) was transferred in a cryovial and immediately stored at -80C until use. A group of 61 occupationally nonexposed white-collar volunteers living in a metropolitan area, of similar age and smoking status as the exposed workers, were also investigated. A similar self-administered questionnaire was used to collect information on personal characteristics and smoking habits. The same biological specimens were obtained as above for the determination of oxidative stress marker levels. Individual written informed consent and approval from the local Institutional Review Board were obtained before the study. Exposure assessment
During their daily activity, workers entered 15 different areas within the plant, with a wide variation of PM exposure concentrations. Particle metal components (cadmium, cobalt, chromium, iron, manganese, lead, tin, zinc, barium, arsenic, nickel, aluminium, copper, strontium, molybdenum, and antimony) and particle mass (PM with aerodynamic diameters
NEWS & VIEWS
Does Enhancement of Oxidative Stress Markers Mediate Health Effects of Ambient Air Particles? Pier Alberto Bertazzi,1 Laura Cantone,1 Pasquale Pignatelli,2 Laura Angelici,1 Valentina Bollati,1 Matteo Bonzini,3 Michele Carugno,1 Pier Mannuccio Mannucci,4 and Francesco Violi 2
Abstract
Evidence indicates that oxidative stress generation may contribute to health effects associated with particulate matter (PM) exposure. We investigated oxidative stress markers in 113 workers exposed to metalrich PM and 61 nonexposed comparable volunteers. The plasma levels of soluble NOX2-derived peptide (sNOX2-dp) and two oxidative stress markers, urinary 8-hydroxydeoxyguanosine (8-OHdG) and 8iso-prostaglandinF2alpha (8-iso-PGF2a), were analyzed. The plasma levels of the antioxidant alphatocopherol were also evaluated. The workers’ average exposure to PM with an aerodynamic diameter < 10 lm (PM10) was much higher at the workplace than in the city, where the volunteers lived. Workers had significantly higher urinary 8-iso-PGF2a and 8-OHdG and plasma sNOX2-dp levels than nonexposed subjects. Alpha-tocopherol was much lower in workers compared with nonexposed subjects. In multivariable regression models adjusted for age, body mass index, and smoking, 8-iso-PGF2a increased in workers in association with PM10 and metal exposure; 8-OHdG and sNOX2-dp were associated only with iron. Alpha-tocopherol was inversely associated with each of the oxidative stress markers. Our observation leads to the hypothesis that the enhancement of oxidative stress markers associated with exposure to high metalrich PM levels represents a possible step in the pathways leading from particle exposure to systemic (e.g., cardiovascular) effects. Antioxid. Redox Signal. 21, 46–51.
Introduction
Innovation
E
pidemiological studies have repeatedly linked environmental exposure to particulate matter (PM) with increased mortality and morbidity from respiratory and cardiovascular disease, in particular, myocardial infarction and ischemic stroke (2). PM metal components have been shown to play key roles in determining PM-related cardiovascular effects. The mechanisms linking the inhalation of air particles to cardiovascular diseases are not completely understood, but PM-induced systemic inflammation has been suggested as a primary mediator. In addition, local and systemic oxidative stress has emerged as a likely common link between pulmonary exposure and systemic effects, including those on the cardiovascular system (2).
Extensive evidence indicates that the generation of oxidative stress contributes to the health effects associated with particulate matter (PM) exposure. We provide evidence that in subjects exposed to high levels of metal-rich PM, the soluble NOX2-derived peptide (sNOX2-dp), urinary 8-hydroxydeoxyguanosine (8-OHdG), and 8-iso-prostaglandinF2alpha (8-isoPGF2a) levels were elevated after controlling for age, smoke, and body mass index, suggesting a role for this enzymatic pathway in reactive oxygen species (ROS) overproduction. In addition, our findings suggest that following the PM stimulus for ROS formation, alpha-tocopherol is lowered as a consequence of its consumption.
1
Department of Clinical Sciences and Community Health, Center of Molecular and Genetic Epidemiology, Universita` di Milano and Fondazione IRCCS Ca` Granda Ospedale Maggiore Policlinico, Milano, Italy. 2 Clinica Medica, Sapienza, University of Rome, Roma, Italy. 3 Department of Clinical and Experimental Medicine, University of Insubria, Varese, Italy. 4 Scientific Direction, Fondazione IRCCS Ca` Granda, Ospedale Maggiore Policlinico, Milan, Italy.
46
OXIDATIVE STRESS, PM, AND CARDIOVASCULAR RISK
PM has been shown to produce oxidative stress in both in vitro and in vivo systems. In vitro studies have demonstrated a clear link between PM actions and levels of reactive oxygen species (ROS), and in vivo studies have consistently found evidence for increased levels of oxidative stress in PM-exposed animals. In rodents, a time- and dose-dependent production of ROS (i.e., superoxide and H2O2) was observed after exposure to concentrated ambient air pollution particles. The components in inhalable PM associated with ROS generation include metals (4). Indoor work conditions may cause a considerably higher exposure to PM than outdoor conditions. For example, in steel plants, workers are exposed to inhalable PM at levels well above the concentrations found in outdoor ambient air, and the PM mixtures contain a large proportion of potentially toxic metal components. In addition, in epidemiological studies, steel workers have been found to be at a higher risk for cardiovascular disease (1). Based on these studies, we selected a steel production plant with a wide range of exposures to well-characterized PM concentrations as a model to investigate whether PM and PM metal components induce changes in the following three oxidative stress markers: the plasma catalytic subunit of NADPH oxidase (NOX2), urinary 8-hydroxydeoxyguanosine (8-OHdG), and urinary isoprostanes (8-iso-PGF2a). Among the various enzymes that generate (intra)cellular ROS, the phagocyte NOX2 has the capacity to transport electrons across the plasma membrane and to generate superoxide and other downstream ROS. An increased NOX2 activity contributes to many health consequences, in particular, cardiovascular disease (7). ROS, produced either endogenously or exogenously, can simultaneously attack lipids, proteins, and nucleic acids in living cells. 8-OHdG, an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. The urinary excretion of 8-OHdG has been often used as a biomarker to assess the extent of repair of ROS-induced DNA damage in both clinical and occupational settings (5). Isoprostanes are prostaglandin-like substances produced in vivo independently of cyclooxygenase enzymes, primarily by free radicalinduced peroxidation of arachidonic acid (8). In particular, F2-isoprostanes are considered the best available biomarkers of oxidative stress status and lipid peroxidation in vivo because other products of the isoprostane pathway, such as D2- and E2-isoprostanes, are less suitable as they are less stable (8). Altogether, substantial evidence has indicated urinary 8-OHdG and isoprostanes as biomarkers of generalized cellular oxidative stress (5). Participants’ characteristics and exposure levels
The mean age of the 113 enrolled workers was comparable to that of nonexposed subjects. The proportion of current smokers and the average body mass index (BMI) did not differ between the two groups. The participants’ characteristics are reported in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/ars). The study workers displayed wide ranges of PM exposure. The highest mean levels of PM-associated metals were measured for zinc, iron, and lead. Details are provided in Supplementary Table S2.
47 Oxidative stress markers and alpha-tocopherol
In exposed workers, urinary 8-OHdG strongly correlated with urinary 8-iso-prostaglandinF2alpha (8-iso-PGF2a) and plasma soluble NOX2-derived peptide (sNOX2-dp). The correlation between urinary 8-iso-PGF2a and plasma sNOX2-dp was also statistically significant. In nonexposed volunteers, these stress oxidative markers were not significantly correlated. Workers had significantly higher urinary 8-iso-PGF2a, urinary 8-OHdG, and plasma sNOX2-dp than nonexposed volunteers. The differences between the oxidative stress markers in the two groups were statistically significant. The plasma levels of alpha-tocopherol were considerably lower in exposed workers compared with nonexposed volunteers. Raw data and statistics are provided in Supplementary Table S3. Association of oxidative stress markers with total PM mass and exposure to metal components
In exposed workers, urinary isoprostanes increased in unadjusted models in association with PM1 and several metal components. The model adjusted for age, BMI, and current smoking status confirmed this observation (Fig. 1). In particular, 8-iso-PGF2a increased in association with PM10, cadmium, cobalt, iron, lead, tin, barium, nickel, copper, and antimony (see Supplementary Data). When the association between urinary 8-OHdG and exposure was examined, an association was observed with iron. Plasma sNOX2-dp was associated with iron and copper (see Supplementary Data). Association of oxidative stress markers with plasma alpha-tocopherol levels
In exposed workers, multivariable regression models indicated that alpha-tocopherol had a significant negative association with urinary 8-OHdG, plasma sNOX2-dp, and urinary 8-iso-PGF2a (Fig. 2). In unexposed subjects, only urinary 8iso-PGF2a displayed a statistically significant negative association with alpha-tocopherol (see Supplementary Data). Discussion
The induction of oxidative stress and inflammation are important mechanisms that mediate the health effects caused by inhaled particles (6). In this study, we examined markers of oxidative stress, such as urinary isoprostanes and 8-OHdG, and found that subjects exposed to high levels of metal-rich PM in an occupational setting demonstrated enhanced oxidative stress compared with subjects living in a metropolitan area with no occupational exposure. This observation indicates that metalrich PM may be a stimulus for enzymatic pathways that ultimately result in ROS formation. To further explore this hypothesis, the circulating levels of NOX2, one of the most relevant cellular producers of ROS, were measured. In this study, we provide the first evidence that the NOX2 activity is increased in subjects exposed to metal-rich air polluting PM, suggesting a role for this enzymatic pathway in ROS overproduction. Several cellular lines may contribute to NOX2 upregulation. The assay used in this study mainly reflects NOX2 activation in blood cells, such as platelets, leucocytes, and monocytes, and it is conceivable that more than one cellular line contribute to NOX2 upregulation and, in turn, to ROS overproduction.
48
BERTAZZI ET AL.
FIG. 1. Multivariable linear regression models adjusted for age, body mass index (BMI), current smoking status (Yes/No). *p-Values £ 0.05. Geometric mean ratio (GMR) and 95% confidence interval (CI) expressing the changes in oxidative stress markers associated with an increase in exposure equal to the difference between the 90th and 10th percentile of the exposure distribution.
Consistent with this idea was the observed correlation in exposed workers for both urinary markers 8-OHdG and isoprostanes with NOX2 plasma levels. The direct association between serum NOX2 and urinary isoprostane is in concordance with previous reports showing that downregulation and upregulation of NOX2 were associated with decreased and enhanced formation of isoprostane, respectively, identifying NOX2 as one of the most important contributors to isoprostane formation (7).
We investigated whether the type and composition of PM affected oxidative stress. Urinary isoprostanes (8-iso-PGF2a) were associated with the finest particles (PM1) and several metal components, in particular, iron. Urinary 8-OHdG and plasma sNOX2-dp were also associated with iron. Iron is essential for metabolic processes and normal cellular functioning. However, increased availability of iron may modulate the production of ROS via the Fenton reaction and induce
FIG. 2. Multivariable linear regression models adjusted for age, BMI, and current smoking status (Yes/No). GMR [ = exp(b)] and 95% CIs. (GMR-1)*100 expresses the percentage change in oxidative stress markers associated with one lmol/mmol chol increase in alpha-tocopherol.
OXIDATIVE STRESS, PM, AND CARDIOVASCULAR RISK
oxidative injury and cellular toxicity. The increase of oxidative stress markers observed in this study may be associated with bioavailable iron from inhaled particle mixtures, suggesting an overload of inhaled iron as a critical toxicant for normal biological processes. New and relevant information stems from the present analysis of plasma levels of alpha-tocopherol, an antioxidant inhibiting lipid peroxidation (9). Alpha-tocopherol/ plasma cholesterol levels, the most reliable method for measuring alpha-tocopherol in blood, were greatly reduced in workers exposed to metal-rich PM and inversely associated with markers of oxidative stress. This observation suggests that upon continuous exposure to air pollution and ensuing stimulation for ROS formation, alpha-tocopherol is lowered as a consequence of its consumption. An intriguing consequence of this argument is the possibility of blunting ROS overproduction by air pollution and its potentially deleterious effect on the human body by alpha-tocopherol supplementation. This study has inherent limitations. Although workers were operating in a modern steel plant with state-of-the-art systems for exposure control, we cannot exclude that exposure to factors other than PM and metals, such as heat, carbon monoxide, and nonionizing radiation, might have contributed to the observed effects (3). Only area and nonpersonal measurements were available for exposure assessment. A further limitation was that unexposed subjects represented a convenience sample because of the known difficulty in obtaining a nonexposed reference sample when conducting studies on working populations. However, the characteristics of the two male samples in terms of age, BMI, tobacco smoking, and health conditions were comparable. Hence, these limitations are unlikely to seriously detract from the validity of our findings. The investigation of mechanisms linking the inhalation of air particles to cardiovascular disease is considered a high priority of biomedical research (2). The exploration of mechanisms involving alterations in oxidative stress markers and alpha-tocopherol in this study may help shed new light on the chain of events that unfold from particle exposure resulting in cardiovascular disease. Our findings could lead to the identification of potentially reversible alterations as potential targets for secondary prevention interventions, including supplementation with alpha-tocopherol. Notes Study population
We recruited 113 healthy active males aged 20 to 60 years, apparently free from disease, who worked in a steel production plant in Italy for at least 1 year. Individual written informed consent and approval from the local Institutional Review Board were obtained before the study. A selfadministered questionnaire was used to collect detailed information on smoking habits, drug use (including nonsteroidal anti-inflammatory drugs (NSAID), underlying medical conditions, BMI, and educational levels. Records from the factory administrative files were used to extract information on occupational history. Biological specimens were collected for each subject, including blood in EDTA tubes and urine morning spot samples. A whole blood aliquot (300 ll) was used for a complete blood count to obtain the absolute number and percentage of
49
granulocytes. The remaining whole blood was centrifuged on site (in an appropriately protected and equipped room in the plant infirmary, within 30 min of blood drawing) at 2500 rpm for 15 min to separate platelet-poor plasma. A plasma aliquot (200 ll) was transferred in a cryovial and immediately stored at -80C until use. A group of 61 occupationally nonexposed white-collar volunteers living in a metropolitan area, of similar age and smoking status as the exposed workers, were also investigated. A similar self-administered questionnaire was used to collect information on personal characteristics and smoking habits. The same biological specimens were obtained as above for the determination of oxidative stress marker levels. Individual written informed consent and approval from the local Institutional Review Board were obtained before the study. Exposure assessment
During their daily activity, workers entered 15 different areas within the plant, with a wide variation of PM exposure concentrations. Particle metal components (cadmium, cobalt, chromium, iron, manganese, lead, tin, zinc, barium, arsenic, nickel, aluminium, copper, strontium, molybdenum, and antimony) and particle mass (PM with aerodynamic diameters
