Article
In vitro effects of exogenous carbon monoxide on oxidative stress and lipid metabolism in macrophages
Toxicology and Industrial Health 1–6 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714558084 tih.sagepub.com
Lauren Petrick1,2, Mira Rosenblat2 and Michael Aviram2 Abstract Carbon monoxide (CO) is a major constituent of traffic-related air pollution and is also produced endogenously under conditions of oxygen-mediated stress. It has been shown to affect both oxidative stress and inflammation. However, its role in lipid metabolism has been neglected. Using short exposure times, the effect of CO on J774A.1 macrophage atherogenic functions was investigated up to 16 h after exposure. Exposure of macrophages was found to be pro-atherogenic as it significantly increased triglyceride mass, up to 60%, and decreased high-density lipoprotein-mediated cholesterol efflux, up to 27%. In contrast, paraoxonase 2 lactonase activity was increased, up to 65%, and cellular oxidative stress was attenuated by 29%, compared with the control cells. The above results on lipid metabolism may lead to arterial macrophage foam cell formation, the hallmark of early atherogenesis. Keywords Carbon monoxide (CO), triglycerides (TG), oxidative stress, paraoxonase 2 (PON2), cholesterol efflux, macrophages
Introduction Primary traffic emissions are a complex mixture of particulate matter with gaseous co-pollutants including nitrogen oxides, hydrocarbons, and carbon monoxide (CO) at relatively high concentrations. In fact, CO emissions can be of the order of 100 ppm (Campen et al., 2010), leading to urban outdoor concentrations ranging from 0.5 ppm to 30 ppm (Delamater et al., 2012; Rubio et al., 2010). CO exposure has been shown to induce markers of vascular remodeling and inflammation in atherosclerotic ApoE/ mice (Campen et al., 2010; Seilkop et al., 2012 ) and induce higher oxidative stress and cardiac arrhythmia in rats (Andre´ et al., 2011). Conversely, endogenous CO has recently been suggested as a possible therapeutic agent with antiatherosclerotic properties. Endogenous production of CO occurs via enzymatic heme degradation by heme oxygenase (HO). The role of HO-1 expression on antiatherogenic properties in animal models has been well established (Liu et al., 2012; Orozco et al., 2007), and CO and CO-releasing molecules have been shown to abate both inflammation and oxidative stress, stressors linked with atherosclerosis formation
(Rochette et al., 2013). While these therapeutic studies focused on CO-induced decrement in oxidative stress and inflammation, they neglected proatherosclerotic factors such as lipid metabolism. Furthermore, whether exogenous CO exposure results in similar overall antiatherogenic properties as endogenous CO exposure remains unclear. Atherosclerosis is characterized by lipid-laden macrophages containing mostly cholesterol esters but also a substantial amount of triglycerides (TGs) within core lipid droplets. In fact, TGs are an independent risk factor for atherosclerosis development (Carmena et al., 2004). The current study seeks to provide additional information regarding the effects of exogenous CO exposure on lipid metabolism in 1
The Technion Center of Excellence in Exposure Science and Environmental Health (TCEEH), Technion, Israel 2 The Lipid Research Laboratory, Rappaport Faculty of Medicine and Research Institute, Technion, Israel Corresponding author: Lauren Petrick, Faculty of Civil and Environmental Engineering, Technion, Haifa, 32000, Israel. Email: [email protected]
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Toxicology and Industrial Health
J774 macrophages, a widely used murine line that is representative of arterial macrophages (Huff et al., 1991).
Methods Chemicals Dihydrocumarin (DHC) and 20 ,70 -dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma Aldrich (St Louis, Missouri, USA). Phosphatebuffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS; heat inactivated at 56 C for 30 min), penicillin, streptomycin, nystatin, L-glutamine and sodium pyruvate were purchased from Biological Industries (Beth Haemek, Israel).
Cell culture and exposure J774A.1 murine macrophage cell line (ATCC, Rockville, Maryland, USA; 1–10 105 mL1) were incubated in DMEM containing 5% FCS and 1% penicillin/streptomycin/nystatin solution at 37 C in humidified air (5% carbon dioxide (CO2)). Medium was aspirated and replaced with medium containing CO or with control medium and incubated overnight in CO-free air/5% CO2. Exposed cells were used for in vitro assays.
CO-infused medium preparation CO with 99.9% purity (Linde, Germany) was diluted to 1000, 10, and 1 ppm in 25 L Teflon bags containing dry air/7% CO2. The CO mixture or dry air/7% CO2 mixture (control) was then bubbled through a sintered glass filter into 100 mL of DMEM containing 5% FCS and 1% penicillin/streptomycin/nystatin solution overnight using a peristaltic pump (10 mL min1). CO2 was required to maintain medium pH ¼ 7.5 while bubbling. This solution was then aliquoted into 10 mL tubes and stored at 4 C to preserve aqueous CO concentrations throughout experiments. Exposure dosages are specified as 1, 10, and 1000 ppm representing the CO/air concentration used for infusing the medium with CO. CO gas concentration in the medium could not be measured due to technical limitations. Although incubation with J774A.1 macrophages was performed overnight, the CO available for exposure was only that dissolved in the medium. Upon introduction to the CO-free incubator air, the CO gradually diffused out of the medium due to the
concentration gradient between the CO in the medium and that in the incubator air (assumed to be negligible). To estimate the exposure time of CO, the diffusion coefficient (D, in square centimeter per second) was determined using the Stokes–Einstein equation (equation (1)) and a transfer coefficient or ‘‘piston velocity’’ (kg; in centimeter per second) determined using equation (2) D¼
RT ; 3NA d
ð1Þ
D ; Z
ð2Þ
kg ¼
where R is the gas constant (8.314 J K1 mmol1), T is temperature (K), NA is Avogadro’s number, is the solution viscosity (kg m1s1), d is the CO molecule diameter (m), and Z is the average diameter of the medium laminar boundary layer through which the diffusion is taking place (cm). Based on the experimental conditions with temperature of 37 C, CO diameter of 113 pm (Demaison and Csa´sza´r, 2012), DMEM viscosity of 7.1 104 Pas (Hinderliter et al., 2010), and Z value of 60 mm, the diffusion coefficient for CO was estimated to be 2.8 105 cm2 s1 and the transfer coefficient estimated to be 4.7 103 cm s1. For a 0.5-cm well liquid thickness, full gas exchange with the incubator took place every 106 s. Thus, the actual exposure time is expected to be of the order of only a few minutes due to CO degassing from the small volume of medium.
In vitro assays Macrophage ROS formation. Cellular reactive oxygen species (ROS) levels were determined using DCFHDA as a probe (LeBel et al., 1992). Cells were washed and incubated with 10 mM DCFH-DA for 30 min at 37 C. Reaction was stopped by PBS washes (2), and cellular fluorescence was measured with a flow cytometry apparatus (FACS-SCAN, Becton Dickinson, San Jose, California, USA). Lipid extraction. Cell lipids were extracted with hexane:isopropanol (3:2, v:v), and the hexane phase evaporated under nitrogen. Cellular TG or cholesterol in dried samples was determined using commercial kits (Sigma, cat no. TR0100, or CHOL, Roche Diagnostics DmbH, Switzerland). Cellular protein was measured after addition of 0.1 N sodium hydroxide with the Lowry assay (Lowry et al., 1951).
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Petrick et al.
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Figure 1. Effect of CO on macrophage TG mass, n ¼ 3; *p:
In vitro effects of exogenous carbon monoxide on oxidative stress and lipid metabolism in macrophages
Toxicology and Industrial Health 1–6 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714558084 tih.sagepub.com
Lauren Petrick1,2, Mira Rosenblat2 and Michael Aviram2 Abstract Carbon monoxide (CO) is a major constituent of traffic-related air pollution and is also produced endogenously under conditions of oxygen-mediated stress. It has been shown to affect both oxidative stress and inflammation. However, its role in lipid metabolism has been neglected. Using short exposure times, the effect of CO on J774A.1 macrophage atherogenic functions was investigated up to 16 h after exposure. Exposure of macrophages was found to be pro-atherogenic as it significantly increased triglyceride mass, up to 60%, and decreased high-density lipoprotein-mediated cholesterol efflux, up to 27%. In contrast, paraoxonase 2 lactonase activity was increased, up to 65%, and cellular oxidative stress was attenuated by 29%, compared with the control cells. The above results on lipid metabolism may lead to arterial macrophage foam cell formation, the hallmark of early atherogenesis. Keywords Carbon monoxide (CO), triglycerides (TG), oxidative stress, paraoxonase 2 (PON2), cholesterol efflux, macrophages
Introduction Primary traffic emissions are a complex mixture of particulate matter with gaseous co-pollutants including nitrogen oxides, hydrocarbons, and carbon monoxide (CO) at relatively high concentrations. In fact, CO emissions can be of the order of 100 ppm (Campen et al., 2010), leading to urban outdoor concentrations ranging from 0.5 ppm to 30 ppm (Delamater et al., 2012; Rubio et al., 2010). CO exposure has been shown to induce markers of vascular remodeling and inflammation in atherosclerotic ApoE/ mice (Campen et al., 2010; Seilkop et al., 2012 ) and induce higher oxidative stress and cardiac arrhythmia in rats (Andre´ et al., 2011). Conversely, endogenous CO has recently been suggested as a possible therapeutic agent with antiatherosclerotic properties. Endogenous production of CO occurs via enzymatic heme degradation by heme oxygenase (HO). The role of HO-1 expression on antiatherogenic properties in animal models has been well established (Liu et al., 2012; Orozco et al., 2007), and CO and CO-releasing molecules have been shown to abate both inflammation and oxidative stress, stressors linked with atherosclerosis formation
(Rochette et al., 2013). While these therapeutic studies focused on CO-induced decrement in oxidative stress and inflammation, they neglected proatherosclerotic factors such as lipid metabolism. Furthermore, whether exogenous CO exposure results in similar overall antiatherogenic properties as endogenous CO exposure remains unclear. Atherosclerosis is characterized by lipid-laden macrophages containing mostly cholesterol esters but also a substantial amount of triglycerides (TGs) within core lipid droplets. In fact, TGs are an independent risk factor for atherosclerosis development (Carmena et al., 2004). The current study seeks to provide additional information regarding the effects of exogenous CO exposure on lipid metabolism in 1
The Technion Center of Excellence in Exposure Science and Environmental Health (TCEEH), Technion, Israel 2 The Lipid Research Laboratory, Rappaport Faculty of Medicine and Research Institute, Technion, Israel Corresponding author: Lauren Petrick, Faculty of Civil and Environmental Engineering, Technion, Haifa, 32000, Israel. Email: [email protected]
Downloaded from tih.sagepub.com at DEAKIN UNIV LIBRARY on November 14, 2015
2
Toxicology and Industrial Health
J774 macrophages, a widely used murine line that is representative of arterial macrophages (Huff et al., 1991).
Methods Chemicals Dihydrocumarin (DHC) and 20 ,70 -dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma Aldrich (St Louis, Missouri, USA). Phosphatebuffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS; heat inactivated at 56 C for 30 min), penicillin, streptomycin, nystatin, L-glutamine and sodium pyruvate were purchased from Biological Industries (Beth Haemek, Israel).
Cell culture and exposure J774A.1 murine macrophage cell line (ATCC, Rockville, Maryland, USA; 1–10 105 mL1) were incubated in DMEM containing 5% FCS and 1% penicillin/streptomycin/nystatin solution at 37 C in humidified air (5% carbon dioxide (CO2)). Medium was aspirated and replaced with medium containing CO or with control medium and incubated overnight in CO-free air/5% CO2. Exposed cells were used for in vitro assays.
CO-infused medium preparation CO with 99.9% purity (Linde, Germany) was diluted to 1000, 10, and 1 ppm in 25 L Teflon bags containing dry air/7% CO2. The CO mixture or dry air/7% CO2 mixture (control) was then bubbled through a sintered glass filter into 100 mL of DMEM containing 5% FCS and 1% penicillin/streptomycin/nystatin solution overnight using a peristaltic pump (10 mL min1). CO2 was required to maintain medium pH ¼ 7.5 while bubbling. This solution was then aliquoted into 10 mL tubes and stored at 4 C to preserve aqueous CO concentrations throughout experiments. Exposure dosages are specified as 1, 10, and 1000 ppm representing the CO/air concentration used for infusing the medium with CO. CO gas concentration in the medium could not be measured due to technical limitations. Although incubation with J774A.1 macrophages was performed overnight, the CO available for exposure was only that dissolved in the medium. Upon introduction to the CO-free incubator air, the CO gradually diffused out of the medium due to the
concentration gradient between the CO in the medium and that in the incubator air (assumed to be negligible). To estimate the exposure time of CO, the diffusion coefficient (D, in square centimeter per second) was determined using the Stokes–Einstein equation (equation (1)) and a transfer coefficient or ‘‘piston velocity’’ (kg; in centimeter per second) determined using equation (2) D¼
RT ; 3NA d
ð1Þ
D ; Z
ð2Þ
kg ¼
where R is the gas constant (8.314 J K1 mmol1), T is temperature (K), NA is Avogadro’s number, is the solution viscosity (kg m1s1), d is the CO molecule diameter (m), and Z is the average diameter of the medium laminar boundary layer through which the diffusion is taking place (cm). Based on the experimental conditions with temperature of 37 C, CO diameter of 113 pm (Demaison and Csa´sza´r, 2012), DMEM viscosity of 7.1 104 Pas (Hinderliter et al., 2010), and Z value of 60 mm, the diffusion coefficient for CO was estimated to be 2.8 105 cm2 s1 and the transfer coefficient estimated to be 4.7 103 cm s1. For a 0.5-cm well liquid thickness, full gas exchange with the incubator took place every 106 s. Thus, the actual exposure time is expected to be of the order of only a few minutes due to CO degassing from the small volume of medium.
In vitro assays Macrophage ROS formation. Cellular reactive oxygen species (ROS) levels were determined using DCFHDA as a probe (LeBel et al., 1992). Cells were washed and incubated with 10 mM DCFH-DA for 30 min at 37 C. Reaction was stopped by PBS washes (2), and cellular fluorescence was measured with a flow cytometry apparatus (FACS-SCAN, Becton Dickinson, San Jose, California, USA). Lipid extraction. Cell lipids were extracted with hexane:isopropanol (3:2, v:v), and the hexane phase evaporated under nitrogen. Cellular TG or cholesterol in dried samples was determined using commercial kits (Sigma, cat no. TR0100, or CHOL, Roche Diagnostics DmbH, Switzerland). Cellular protein was measured after addition of 0.1 N sodium hydroxide with the Lowry assay (Lowry et al., 1951).
Downloaded from tih.sagepub.com at DEAKIN UNIV LIBRARY on November 14, 2015
Petrick et al.
3
Figure 1. Effect of CO on macrophage TG mass, n ¼ 3; *p:
