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Oxyfuel in Alaska: use of interleukins to monitor effects on the immune system
the Science of the Total Environment ELSEVIER
The Science of the Total Environment 151 (1994) 253-256
Short communication
Oxyfuel in Alaska: use of interleukins to monitor effects on the immune system Lawrence K. Duffy* Department of Chemistry and Institute of Arctic Biology, Universityof Alaska Fairban~, Fairbanks, Alaska 99775, USA Received 9 July 1993; accepted 20 July 1993
Abstract
During a 4-week period in late November and early December 1992, blood samples from individuals exposed to auto emissions derived from oxyfuel were analyzed. Effects on the immune system were measured by monitoring plasma interleukin 6 (IL-6) levels in a total of 22 subjects at the beginning and the end of the workday. After ~ 8 h of workplace exposure, mean levels of IL-6 of 2.5 pg/ml were obtained. While some subjects showed increasing levels at the end of the workday, there was no difference between morning and evening IL-6 means. Our conclusion is that single day exposures to oxyfuel and its combustion products does not show an immediate effect on the immune system as judged by serum IL-6 levels.
Keywords: Interleukins, Methyl ten-butyl ether (MTBE), Oxyfuel, Fairbanks, Alaska (65°N)
I. Introduction The US Environmental Protection Agency (EPA) establishes national ambient air quality standards for air pollutants as well as emission standards for hazardous pollutants. Because carbon monoxide (CO) levels in Fairbanks exceeded standards in 1991, for the winter of 1992-1993, the EPA required that automobiles in Fairbanks and Anchorage, Alaska, use oxygenated fuel from November to March. The use of a gasoline addi-
* Corresponding author.
tive - - methyl tertiary butyl ether (MTBE) - - at laboratory temperatures reduces the emission of carbon monoxide, but increases the emission of various aldehydes, including formaldehyde, by ~ 1 part per million (ppm) [1]. Oxygenated fuel, which is 85% gasoline and 15% MTBE, has been reported to lead to exposures of < 0.4 ppm formaldehyde, the EPA standard is 1 ppm. The effect of oxygenated fuels on the reduction of CO levels is still undergoing evaluation [2]. Other pollutants which are associated with oxygenated fuels are formic acid and peroxyacetyl nitrate (PAN), a mutagenic compound that is a severe eye irritant.
0048-9697/94/$07.00 © 1994 Elsevier Science BV. All rights reserved.
SSD1 0048-9697(94)04019-6
254
L.K Duffy / Sci. Total En~iron. 151 (1994) 253 256
After the introduction of oxyfuels to Fairbanks, ~ 30% of the 200 residents, interviewed by doctors from the Center for Disease Control (CDC), reported symptoms which included headaches, nausea and throat and eye irritation [3]. Few tests have been conducted on the health aspects surrounding oxygenated fuels. These symptoms are similar to those associated with formaldehyde toxicity [4]. Also, subacute effects on the immune system are possible. Tests on mice showed a slight increase in natural killer cell-mediated cytotoxicity and in rats showed increased counts of white blood cells [5]. Similarly, oxidant injury from ozone has been shown to affect alveolar marcrophagederived paracrine factors [5-7], and leads to the inhibition of several intercellular enzymes and the depletion of the intracellular glutathione pool [8,9]. Tanswell et al. [10,11] have presented evidence for the production of paracrine and autocrine factors after injury. Although these factors have not been completely identified, they have the general properties of cytokines. Cytokines are a group of proteins of low molecular weight (usually < 20 Kd) used by immune and injured cells to communicate with each other. They take part in all phases of injury, including inflammation, immune response, hemopoiesis and repair of tissue. Cytokines are produced at a site of injury or stimulation and act locally in a paracine or autocrine fashion. They may also circulate, and have hormonal-like effects - - acting at sites distant from the original site of secretion [12]. The hypothesis underlying this study is that cellular injury due to MTBE exposure will result in the local generation of cytokines and that these should leak into the blood stream. Such factors could be used as early markers of lung injury. Since Fairbanks is at 65°N and the immune system is affected by seasonal light patterns [13], we focused on the effect of daily oxyfuel exposure on the well characterized cytokines, interleukin 6 (IL-6) and interleukin 1/3 (IL-1/3).
40 30 " o...
20
bJ
r,-
10
a:
0
ZD
~ -10 w F--
oI_ _
VLr,
30
I÷
-4O
l i
I0
20 NOV
30
I0
20 DEC
30
Fig. 1. Average daily temperatures in Fairbanks, Alaska, during November and December 1992. Normal daily activities, which included indoor duties as mechanics, were maintained. Blood samples were taken at the beginning of shifts (6-8 am) and at the end of the workday (4-6 pm). Samples received from CDC doctors were analyzed for IL-6 levels using an immunochemical assay (Quantakine ELISA, R and D, Inc. Minneapolis, MN). Samples, which were run in duplicate, were added to a microliter plate coated with a monoclonal antibody for IL-6. After washing away any unbound proteins, an enzyme-linked polyclonal antibody for IL-6 was added to the wells and incubated to allow for any IL-6 binding. After a final wash, a substrate solution was added to the wells. After color develops, sample concentrations were determined from a standard curve. IL-1/3 was measured similarly. 3. Results and discussion
2. Subjects and methods
Twenty-two volunteers at several different locations around Fairbanks participated in the study.
The mean daily temperature for Fairbanks, Alaska (65°N) is shown in Fig. 1. During the period oxyfuels were in use, the mean tempera-
L.K. Duffy / Sci. Total Entqron. 151 (1994) 253-256
80[
255
8° I 70
7O 10
10
-r ~- - 8
8
E
E ---6
6 LO
oo
_3
4
I--.,I
(2.50 1 2 4 )
T
i! 0
II,
4
',2.53 + 2.6)
$ |
2
N
AM
PM
Fig. 2. Range of subject response in IL-6 after workplace exposure to M T B E fuels. The circles denote individual levels and the squares denote the m e a n level for all subjects ( _+S.D.).
ture ranged from 35°F to - 3 8 ° F . Another important variable is daylight, which varies from ~ 5 h in November to 3 h 45 min in the middle of December [13]. IL-1, which was measured in 10 subjects, was below the detectable limits of the assay, 10 pg/ml. However, IL-6 levels were detected in all but two of the morning samples and five of the evening samples (Fig. 2). There was no difference between the morning mean levels of IL-6 (2.50 _+ 2.4 S.D.) and the evening mean levels (2.53_+ 2.6 S.D.). When the direction of change in IL-6 was analyzed (Fig. 3) a slight increasing trend was observed. Fourteen out of 22 samples showed some increase of IL-6 during the day. Our findings that IL-1 levels are below assay detection limits is in agreement with a previous study on an Anchorage, Alaska, population of recovering alcoholics. When IL-6 levels were measured in the Anchorage study, which was in
0 AM
PM
Fig. 3. Change in IL-6 levels as a mediator of inflammation and an indicator of lung damage after workplace exposure to MTBE. Morning samples were collected at the start of a shift ( 6 - 8 am) and at the end of the workday (4-6 pm).
the year preceding the introduction of oxyfuel in Alaska, 15 out of 38 samples showed detectable IL-6 levels. In a recent report, Devlin et al. [5] measured the IL-1 and IL-6 levels in bronchalveolar lavage (BAL) fluid in humans exposed to ozone for 6.6 h. Although IL-1 was detected in the BAL fluid, there was no change following ozone exposure. However, there was a 393% increase in IL-6 activity in the BAL fluid after exposure to 0.1 ppm ozone. These results conflict with our results on plasma IL-6. This may be due to the lack of any effect of M T B E on macrophages or that any high concentrations seen in tissue would be diluted down to the level of background noise in the serum assay. Although Devlin et al. [5] suggested that inflammation caused by pollutants might be monitored with less invasive techniques
256
L.K. Duffy / Sci. Total Entxron. 151 (1994) 253-256
than BAL, our results indicate that s e r u m analysis may not be effective for low level chronic exoosure. IL-6 is involved in the differentiation of B-cells to IgG-secreting plasma cells as well as an inducer of acute phase proteins a n d cytotoxie T-cells [14]. A n y injury caused to the lung tissue by M T B E , which would increase IL-6 levels in B A L fluid, is unlikely to cause a systemic response since m e a n levels in the s e r u m did not increase. A l t h o u g h there was n o m e a n change in IL-6 levels, there was a considerable n u m b e r of individuals who showed slight increases. In future studies, a follow-up on those individuals for several days may show an effect not observed in this short study.
5
6
7
8
9
Acknowledgements
10
I would like to t h a n k Ms. A n d r e e Porchet for technical assistance. This work was f u n d e d in part by the Institute of Arctic Biology.
11
References 12 1 L.G. Anderson, Tucson aldehyde study 1990-1991, Rep. to Arizona Dept. of Environ. Qual. 2 D. Illman, Oxygenated fuel cost may outweigh effectiveness, Chem. Eng. News, 71, (1993) 28-30. 3 M.B. Belier and J. Middaugh, Evaluation of potential illness due to exposure to oxygenated fuels in Fairbanks, Alaska, Alaska Dept. of Health and Soc. Serv. Rep., Alaska, 1992. 4 S.E. Feinman, Formaldehyde toxicity, in S.E. Feinman
13
14
(Ed), Formaldehyde sensitivity and toxicity, CRC Press, 1988, pp. 155-166. R.B. Devlin, W.F. McDonnell, R. Mann, S. Becker, D.E. House, D. Schreinemackers and H.S. Koren, Exposure of humans to ambient levels for 6.6 h causes cellular and biochemical changes in the lung, Am. J. Respir. Cell Mol. Biol., 4 (1991) 72-81. J.A. Elias, M.D. Rossman, R.B. Zuririer and R.P Daniele, Human alveolar macrophage inhibition of lung fibroblast growth: a prostaglandin-dependent process, Am. Rev. Respir. Dis., 131 (1985) 94-99. P.B. Bitterman, S.I. Rennard, G.W. Hunningbake and R.G. Crystal, Human alveolar macrophage growth factor for fibroblasts: regulation and partial characterization, J. Clin. Invest., 70 (1982) 806-822. B.A. Freeman and J.B. Mudd, Reaction of ozone with human erythrocyte sulfhydryls,Arch. Biochem. Biophys., 208 (1981) 212-220. J. Van der Zee, T.M. Dubbleman, T.K. Raap and J. Van Steveninick, Toxic effect of ozone on murine L929 fibroblasts, Biochem. J., 242 (1987) 707-712. B.A. Freeman and A.K. Tanswell, Biochemical and cellular aspects of pulmonary oxygen toxicity, Adv. Free Radical Biol. Med., 1 (1985) 133-164. A.K. Tanswell, L.J. Fraker and E.C. Grose, Circulating factors which modify lung cell DNA synthesis following exposure to inhaled oxidants: effect of serum and lavage on lung fibroblasts following exposure of rats to 1 ppm ozone, J. Toxicol. Environ. Health, 27 (1989) 239-254. M.L. Rodrick, Cytokine response to infection, in A.F. Roche (Ed), Cytokines in critical illness, Rep. of the llth Ross Conf. on Med. Res., Columbus, Ross Laboratories, Ohio, 1992, pp. 23-25. M.E. Levine, A.N. Milliron and L.K. Duffy, Diurnal and seasonal rhythms of melatonin, cortisol and testosterone in interior Alaska, Arct. Med. Res., 53 (1994) 25-34. T. Kiskimoto and T. Hirano, Molecular regulation of /3 lymphocyte response, Ann. Rev. Immunol., 6 (1988) 485-512.
The Science of the Total Environment 151 (1994) 253-256
Short communication
Oxyfuel in Alaska: use of interleukins to monitor effects on the immune system Lawrence K. Duffy* Department of Chemistry and Institute of Arctic Biology, Universityof Alaska Fairban~, Fairbanks, Alaska 99775, USA Received 9 July 1993; accepted 20 July 1993
Abstract
During a 4-week period in late November and early December 1992, blood samples from individuals exposed to auto emissions derived from oxyfuel were analyzed. Effects on the immune system were measured by monitoring plasma interleukin 6 (IL-6) levels in a total of 22 subjects at the beginning and the end of the workday. After ~ 8 h of workplace exposure, mean levels of IL-6 of 2.5 pg/ml were obtained. While some subjects showed increasing levels at the end of the workday, there was no difference between morning and evening IL-6 means. Our conclusion is that single day exposures to oxyfuel and its combustion products does not show an immediate effect on the immune system as judged by serum IL-6 levels.
Keywords: Interleukins, Methyl ten-butyl ether (MTBE), Oxyfuel, Fairbanks, Alaska (65°N)
I. Introduction The US Environmental Protection Agency (EPA) establishes national ambient air quality standards for air pollutants as well as emission standards for hazardous pollutants. Because carbon monoxide (CO) levels in Fairbanks exceeded standards in 1991, for the winter of 1992-1993, the EPA required that automobiles in Fairbanks and Anchorage, Alaska, use oxygenated fuel from November to March. The use of a gasoline addi-
* Corresponding author.
tive - - methyl tertiary butyl ether (MTBE) - - at laboratory temperatures reduces the emission of carbon monoxide, but increases the emission of various aldehydes, including formaldehyde, by ~ 1 part per million (ppm) [1]. Oxygenated fuel, which is 85% gasoline and 15% MTBE, has been reported to lead to exposures of < 0.4 ppm formaldehyde, the EPA standard is 1 ppm. The effect of oxygenated fuels on the reduction of CO levels is still undergoing evaluation [2]. Other pollutants which are associated with oxygenated fuels are formic acid and peroxyacetyl nitrate (PAN), a mutagenic compound that is a severe eye irritant.
0048-9697/94/$07.00 © 1994 Elsevier Science BV. All rights reserved.
SSD1 0048-9697(94)04019-6
254
L.K Duffy / Sci. Total En~iron. 151 (1994) 253 256
After the introduction of oxyfuels to Fairbanks, ~ 30% of the 200 residents, interviewed by doctors from the Center for Disease Control (CDC), reported symptoms which included headaches, nausea and throat and eye irritation [3]. Few tests have been conducted on the health aspects surrounding oxygenated fuels. These symptoms are similar to those associated with formaldehyde toxicity [4]. Also, subacute effects on the immune system are possible. Tests on mice showed a slight increase in natural killer cell-mediated cytotoxicity and in rats showed increased counts of white blood cells [5]. Similarly, oxidant injury from ozone has been shown to affect alveolar marcrophagederived paracrine factors [5-7], and leads to the inhibition of several intercellular enzymes and the depletion of the intracellular glutathione pool [8,9]. Tanswell et al. [10,11] have presented evidence for the production of paracrine and autocrine factors after injury. Although these factors have not been completely identified, they have the general properties of cytokines. Cytokines are a group of proteins of low molecular weight (usually < 20 Kd) used by immune and injured cells to communicate with each other. They take part in all phases of injury, including inflammation, immune response, hemopoiesis and repair of tissue. Cytokines are produced at a site of injury or stimulation and act locally in a paracine or autocrine fashion. They may also circulate, and have hormonal-like effects - - acting at sites distant from the original site of secretion [12]. The hypothesis underlying this study is that cellular injury due to MTBE exposure will result in the local generation of cytokines and that these should leak into the blood stream. Such factors could be used as early markers of lung injury. Since Fairbanks is at 65°N and the immune system is affected by seasonal light patterns [13], we focused on the effect of daily oxyfuel exposure on the well characterized cytokines, interleukin 6 (IL-6) and interleukin 1/3 (IL-1/3).
40 30 " o...
20
bJ
r,-
10
a:
0
ZD
~ -10 w F--
oI_ _
VLr,
30
I÷
-4O
l i
I0
20 NOV
30
I0
20 DEC
30
Fig. 1. Average daily temperatures in Fairbanks, Alaska, during November and December 1992. Normal daily activities, which included indoor duties as mechanics, were maintained. Blood samples were taken at the beginning of shifts (6-8 am) and at the end of the workday (4-6 pm). Samples received from CDC doctors were analyzed for IL-6 levels using an immunochemical assay (Quantakine ELISA, R and D, Inc. Minneapolis, MN). Samples, which were run in duplicate, were added to a microliter plate coated with a monoclonal antibody for IL-6. After washing away any unbound proteins, an enzyme-linked polyclonal antibody for IL-6 was added to the wells and incubated to allow for any IL-6 binding. After a final wash, a substrate solution was added to the wells. After color develops, sample concentrations were determined from a standard curve. IL-1/3 was measured similarly. 3. Results and discussion
2. Subjects and methods
Twenty-two volunteers at several different locations around Fairbanks participated in the study.
The mean daily temperature for Fairbanks, Alaska (65°N) is shown in Fig. 1. During the period oxyfuels were in use, the mean tempera-
L.K. Duffy / Sci. Total Entqron. 151 (1994) 253-256
80[
255
8° I 70
7O 10
10
-r ~- - 8
8
E
E ---6
6 LO
oo
_3
4
I--.,I
(2.50 1 2 4 )
T
i! 0
II,
4
',2.53 + 2.6)
$ |
2
N
AM
PM
Fig. 2. Range of subject response in IL-6 after workplace exposure to M T B E fuels. The circles denote individual levels and the squares denote the m e a n level for all subjects ( _+S.D.).
ture ranged from 35°F to - 3 8 ° F . Another important variable is daylight, which varies from ~ 5 h in November to 3 h 45 min in the middle of December [13]. IL-1, which was measured in 10 subjects, was below the detectable limits of the assay, 10 pg/ml. However, IL-6 levels were detected in all but two of the morning samples and five of the evening samples (Fig. 2). There was no difference between the morning mean levels of IL-6 (2.50 _+ 2.4 S.D.) and the evening mean levels (2.53_+ 2.6 S.D.). When the direction of change in IL-6 was analyzed (Fig. 3) a slight increasing trend was observed. Fourteen out of 22 samples showed some increase of IL-6 during the day. Our findings that IL-1 levels are below assay detection limits is in agreement with a previous study on an Anchorage, Alaska, population of recovering alcoholics. When IL-6 levels were measured in the Anchorage study, which was in
0 AM
PM
Fig. 3. Change in IL-6 levels as a mediator of inflammation and an indicator of lung damage after workplace exposure to MTBE. Morning samples were collected at the start of a shift ( 6 - 8 am) and at the end of the workday (4-6 pm).
the year preceding the introduction of oxyfuel in Alaska, 15 out of 38 samples showed detectable IL-6 levels. In a recent report, Devlin et al. [5] measured the IL-1 and IL-6 levels in bronchalveolar lavage (BAL) fluid in humans exposed to ozone for 6.6 h. Although IL-1 was detected in the BAL fluid, there was no change following ozone exposure. However, there was a 393% increase in IL-6 activity in the BAL fluid after exposure to 0.1 ppm ozone. These results conflict with our results on plasma IL-6. This may be due to the lack of any effect of M T B E on macrophages or that any high concentrations seen in tissue would be diluted down to the level of background noise in the serum assay. Although Devlin et al. [5] suggested that inflammation caused by pollutants might be monitored with less invasive techniques
256
L.K. Duffy / Sci. Total Entxron. 151 (1994) 253-256
than BAL, our results indicate that s e r u m analysis may not be effective for low level chronic exoosure. IL-6 is involved in the differentiation of B-cells to IgG-secreting plasma cells as well as an inducer of acute phase proteins a n d cytotoxie T-cells [14]. A n y injury caused to the lung tissue by M T B E , which would increase IL-6 levels in B A L fluid, is unlikely to cause a systemic response since m e a n levels in the s e r u m did not increase. A l t h o u g h there was n o m e a n change in IL-6 levels, there was a considerable n u m b e r of individuals who showed slight increases. In future studies, a follow-up on those individuals for several days may show an effect not observed in this short study.
5
6
7
8
9
Acknowledgements
10
I would like to t h a n k Ms. A n d r e e Porchet for technical assistance. This work was f u n d e d in part by the Institute of Arctic Biology.
11
References 12 1 L.G. Anderson, Tucson aldehyde study 1990-1991, Rep. to Arizona Dept. of Environ. Qual. 2 D. Illman, Oxygenated fuel cost may outweigh effectiveness, Chem. Eng. News, 71, (1993) 28-30. 3 M.B. Belier and J. Middaugh, Evaluation of potential illness due to exposure to oxygenated fuels in Fairbanks, Alaska, Alaska Dept. of Health and Soc. Serv. Rep., Alaska, 1992. 4 S.E. Feinman, Formaldehyde toxicity, in S.E. Feinman
13
14
(Ed), Formaldehyde sensitivity and toxicity, CRC Press, 1988, pp. 155-166. R.B. Devlin, W.F. McDonnell, R. Mann, S. Becker, D.E. House, D. Schreinemackers and H.S. Koren, Exposure of humans to ambient levels for 6.6 h causes cellular and biochemical changes in the lung, Am. J. Respir. Cell Mol. Biol., 4 (1991) 72-81. J.A. Elias, M.D. Rossman, R.B. Zuririer and R.P Daniele, Human alveolar macrophage inhibition of lung fibroblast growth: a prostaglandin-dependent process, Am. Rev. Respir. Dis., 131 (1985) 94-99. P.B. Bitterman, S.I. Rennard, G.W. Hunningbake and R.G. Crystal, Human alveolar macrophage growth factor for fibroblasts: regulation and partial characterization, J. Clin. Invest., 70 (1982) 806-822. B.A. Freeman and J.B. Mudd, Reaction of ozone with human erythrocyte sulfhydryls,Arch. Biochem. Biophys., 208 (1981) 212-220. J. Van der Zee, T.M. Dubbleman, T.K. Raap and J. Van Steveninick, Toxic effect of ozone on murine L929 fibroblasts, Biochem. J., 242 (1987) 707-712. B.A. Freeman and A.K. Tanswell, Biochemical and cellular aspects of pulmonary oxygen toxicity, Adv. Free Radical Biol. Med., 1 (1985) 133-164. A.K. Tanswell, L.J. Fraker and E.C. Grose, Circulating factors which modify lung cell DNA synthesis following exposure to inhaled oxidants: effect of serum and lavage on lung fibroblasts following exposure of rats to 1 ppm ozone, J. Toxicol. Environ. Health, 27 (1989) 239-254. M.L. Rodrick, Cytokine response to infection, in A.F. Roche (Ed), Cytokines in critical illness, Rep. of the llth Ross Conf. on Med. Res., Columbus, Ross Laboratories, Ohio, 1992, pp. 23-25. M.E. Levine, A.N. Milliron and L.K. Duffy, Diurnal and seasonal rhythms of melatonin, cortisol and testosterone in interior Alaska, Arct. Med. Res., 53 (1994) 25-34. T. Kiskimoto and T. Hirano, Molecular regulation of /3 lymphocyte response, Ann. Rev. Immunol., 6 (1988) 485-512.