urinary prostacyclin in nonsmokers living with smokers

URINARY PROSTACYCLIN IN NONSMOKERS LIVING WITH SMOKERS Carr J. Smith, Walter T. Morgan, David J. Doolittle Environmental and Molecular Toxicology, Research and Development, Bowman Gray Technical Center, R. J. Reynolds Tobacco Company, Winston-Salem, North Carolina, USA

Thomas H. Fischer Department of Medicine, Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Thomas Ruppert, Gerhard Scherer Analytisch-biologisches Forschungslabor, Munich, Germany This study tested the hypothesis that environmental tobacco smoke ( ETS) exposure increases platelet activation. The concentration of the stable urinary metabolites of thromboxane ( Tx-M) and prostacyclin ( PGI-M) was measured in 3 groups of subjects: 27 nonsmokers who did not live with a smoker, 21 nonsmokers who lived with at least 1 smoker, and 10 cigarette smokers who served as a positive control group. Urinary concentrations of the stable metabolites Tx-M and PGI-M did not differ between the nonsmokers living with at least one smoker and the nonsmokers not living with a smoker ( p = .39 for Tx-M; p = .42 for PGI-M) . There was a statistically significant increase in the urinary concentration of PGI-M in the cigarette smokers when compared with either the nonsmokers living with at least one smoker ( p < .05) or the nonsmokers not living with a smoker ( p < .05). There was no significant difference in Tx-M excretion in the cigarette smokers relative to either nonsmokers living with a smoker ( p = .99) or to nonsmokers in nonsmoking households ( p = .53). In the 21 nonsmokers living with smokers, ETS exposure did not result in platelet activation as measured by either thromboxane or prostacyclin release.

Thromboxanes are synthesized in platelets and can cause vasoconstriction and platelet aggregation after release (Hamberg & Samuelsson, 1974). Prostacyclins are potent inhibitors of platelet aggregation that are produced by blood vessel walls (DeWitt et al., 1983). Therefore, thromboxanes and prostacyclins are considered to be antagonistic in their actions (Mayes, 1985). Several groups have previously shown that an increased urinary level of the thromboxane metabolite 2,3-dinor-thromboxane B2 (TxM) is a marker of in vivo platelet activation and that an increased urinary level of the prostacyclin metabolite 2,3-dinor-6-keto-prostaglandin F1a Received 6 November 1997; accepted 15 December 1997. Address correspondence to Carr J. Smith, PhD, DABT, Bowman Gray Technical Center, R. J. Reynolds Tobacco Company, Winston-Salem, NC 27102, USA. E-mail: [email protected]. 431 Inhalation Toxicology, 10:431–441, 1998 Copyright © 1998 Taylor & Francis 0895–8378/98 $12.00 + .00

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(PGI-M) potentially indicates “activated platelet”–vessel wall interaction (Murray et al., 1985; Nowak et al., 1987; Lassila et al., 1988; Rangemark & Wennmalm, 1991; Dotevall et al., 1992). These groups have reported that cigarette smokers sometimes excrete higher levels of these stable metabolites of prostacyclin and thromboxane. Recently, Glantz and Parmley (1995) reviewed the literature and concluded that “Nonsmokers exposed to secondhand smoke in everyday life exhibit an increased risk of both fatal and nonfatal cardiac events.” Based upon several reports in the literature (Pittilo et al., 1982; Sinzinger & Kefalides, 1982; Burghuber et al., 1986; Davis et al., 1989; Sinzinger & Virgolini, 1989; Steinberg, 1989), Glantz and Parmley hypothesized that exposure of nonsmokers to environmental tobacco smoke (ETS) “increases platelet activity,” thereby conferring increased risk of thrombosis and atherosclerosis. In this study, we tested the hypothesis that ETS exposure is associated with increased platelet activation by measuring the urinary concentration of the stable metabolites of thromboxane and prostacyclin in three groups of subjects. The 3 groups were 27 nonsmokers who did not live with a smoker, 21 nonsmokers who lived with one or more smokers, and 10 smokers who served as a positive control group (Tables 1–3). METHODS Subjects Three different groups of subjects were studied: 27 nonsmokers who did not live with a smoker (13 males, mean age 31.7 ± 16.4 yr; 14 TABLE 1. Individual smoker data

Subject number 1 2 3 4 5 6 7 8 9 10 Mean ± SD a

Gender

Age (yr)

Home

F M F M M F M M M F

32 45 41 57 40 34 42 49 20 42

2 1 1 2 1 1 1 2 2 2

a

Cigarettes/ day

Tx-Mb (pg/mg creatinine)

PGI-Mc (pg/mg creatinine)

SCOT1d (ng/ml)

38.8 39.1 6.9 19.6 16.5 8.9 26.5 51.7 15.9 19.8

47.1 160.9 72.3 63.1 61.7 77.5 74.9 165.5 97.8 60.2

93 169 96 52 115 112 94 155 98 70

644 869 207 477 266 198 363 469 259 691

105 ± 35

444 ± 229

24.4 ± 14.5

88.1 ± 41.8

Home: number of smokers in household. Tx-M: 2,3-dinor-thromboxane B2. c PGI-M: 2,3-dinor-6-keto-prostaglandin F1a . d SCOT1: salivary cotinine sample 1 (day 1); SCOT2: salivary cotinine sample 2 (day 8). b

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females, mean age 29.1 ± 15.1 yr; both genders, mean age 30.3 ± 15.5 yr); 21 nonsmokers who lived with at least 1 smoker (9 males, mean age 23.7 ± 20.1 yr; 12 females, mean age 30.6 ± 18.8 yr; both genders, mean age 27.6 ± 19.2 yr; cigarette smokers (6 males, mean age 42.2 ± 12.4 yr; 4 females, mean age 37.3 ± 5.0 yr; both genders, mean age 40.2 ± 10.0 yr). All subjects were residents of the city or suburbs of Munich, Germany. Subjects continued their normal lifestyle and diet throughout the study. Food intake was not monitored in the study. Only those subjects who did not take any drugs on a regular basis were included. The study was reviewed by the ethics committee of Analytisch-biologisches Forschungslabor and was therefore performed in accordance with the ethical standards of the 1964 Declaration of Helsinki. All persons gave their informed consent prior to their inclusion in the study. Study Design Twenty homes inhabited by at least one smoker and 10 homes without a smoking resident were selected for the 8-day study. Spot urine samples were collected at the end of work day 1 (7 p.m.) and at the end of work day 8 (7 p.m.) of the study. Urine samples were stored at –20°C until assayed. ETS Exposure Assessment Exposure to ETS during the 8-day study period was determined by personal samplers for nicotine and by questionnaires subdivided by hour for each study day (Tables 2 and 3). In addition, urinary and salivary cotinine and creatinine were assessed at the beginning of day 1 and at the

SCOT2 (ng/ml)

UCOT1e (ng/ml)

UCOT2 (ng/ml)

CREAT1f (mg/dl)

CREAT2 (mg/dl)

COT/CREAT1g (µg/g creatinine)

COT/CREAT2 (µg/g creatinine)

718 955 112 388 236 209 266 382 380 262

1810 5491 1462 2652 860 40 2181 4486 2801 7430

4191 5780 2289 2900 2165 3198 2221 3888 3284 2611

43 91 60 128 24 44 88 97 54 114

67.3 143.8 166.1 170.1 77.9 170.7 92.3 144.7 108.8 80.2

4209 6034 2457 2072 1946 2844 2467 4606 5177 6518

6227 4019 1378 1705 2779 1873 2406 2687 3018 3256

391 ± 257

2921 ± 2559

3253 ± 1125

122.2 ± 41.3

3833 ± 1700

2935 ± 1397

e

74 ± 34

UCOT1: urinary cotinine sample 1 (day 1); UCOT2: urinary cotinine sample 2 (day 8). CREAT1: creatinine sample 1 (day 1); CREAT2: creatinine sample 2 (day 8). g COT/CREAT1: cotinine/creatinine sample 1 (day 1); COT/CREAT2: cotinine/creatinine sample 2 (day 8). f

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TABLE 2. Individual nonsmoker data: Smoking household

ETSH (h/day)

NIPS 3 (µg/m )

Tx-Md (pg/mg creatinine)

2.9 3.6 4.6 1.8 3.8 1.6 1 3.3 1.9 1.5 0 2.3 3.9 1.3 3.5 2.8 2.8 2.1 3.9 3 2.9

1 0.6 0.3 0.1 0.3 0.1 0.4 0.3 0.3 0.2 4 0.1 0.4 0.1 0.1 0.3 0.2 0.2 0.3 4.6 1.1

144 65 91 55 94 110 49 73 80 113 36 69 51 79 81 88 83 109 45 95 55

2.6 ± 1.1

0.7 ± 1.2

b

Subject number

Gender

Age (yr)

Home

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

M F M F F M M M M F F M F M F F F F F F M

62 58 11 54 28 6 42 43 9 4 55 11 14 11 40 6 36 34 18 20 18

1 1 2 2 1 1 1 1 1 1 2 1 2 2 1 1 1 1 2 3 3

a

Mean ± SD

c

80 ± 26

PGI-Me (pg/mg creatinine) 89 66 99 70 56 93 74 48 58 130 59 59 59 64 67 90 80 58 84 52 88 73 ± 19

a

Home: number of smokers in household. ETSH: self-reported ETS exposure duration. c NIPS: nicotine on personal sampler. d Tx-M: 2,3-dinor-thromboxane B2. e PGI-M: 2,3-dinor-6-keto-prostaglandin F1a . b

end of day 8. The numerical averages of the day 1 and day 8 cotinine and creatinine values are presented in Tables 1, 2, and 3. Nicotine was monitored using a single-face passive diffusion sampler, which employed a Teflon membrane filter as windscreen and a sodium bisulfate-treated glass-fiber filter as collection medium (Ogden & Maiolo, 1992). Nicotine determination was performed as described by Ogden and Maiolo (1992). A radioimmunoassay (RIA) (Langone et al., 1973) with modification by Haley et al. (1983) was used to measure cotinine in saliva and urine. Urinary creatinine was determined by the Jaffe method using a commercial test kit (Merck AG, Darmstadt, Germany). Tx-M and PGI-M Analyses Radioimmunoassay analysis of the stable urinary metabolites of thromboxane (Tx-M) (Kuhn et al., 1993) and prostacyclin (PGI-M) (Demers et al., 1981) was performed without knowledge of study design or subject group


SCOT1 (ng/ml)

f

g

435

h

CREAT2 (mg/dl)

COT/CREAT1i (µg/g creatinine)


SCOT2 (ng/ml)

UCOT1 (ng/ml)

UCOT2 (ng/ml)

CREAT1 (mg/dl)

1 2 3 1 0 0 0 0 1 1 7 0 0 0 1 1 1 1 2 3 5

2 2 — 0 0 0 0 0 0 2 5 0 0 0 1 1 1 0 2 4 6

6 13 18 9 20 12 8 26 36 30 33 3 9 17 16 4 5 13 29 59 45

10 29 — 9 10 6 15 21 32 28 39 5 14 11 15 6 12 9 8 87 35

243 65 100 86 268 136 57 188 143 123 61 130 70 137 180 116 78 130 138 199 95

137 222 — 211 143 91 108 185 227 87 84 228 168 152 109 97 202 255 37 232 51

2.3 20.0 17.5 9.9 7.5 8.4 13.2 13.6 24.8 24.4 54.4 2.3 12.2 12.4 8.6 3.5 6.4 7.2 21.0 29.7 47.5

7.3 13.1 — 4.0 6.6 6.6 13.5 11.4 13.9 32.1 43.4 2.2 8.0 6.9 13.8 6.2 5.9 3.5 20.4 37.5 68.2

1±2

1±2

20 ± 14

20 ± 19

131 ± 57

151 ± 66

16.5 ± 13.5

16.2 ± 16.8

f

SCOT1: salivary cotinine sample 1 (day 1); SCOT2: salivary cotinine sample 2 (day 8). UCOT1: urinary cotinine sample 1 (day 1); UCOT2: urinary cotinine sample 2 (day 8). h CREAT1: creatinine sample 1 (day 1); CREAT2: creatinine sample 2 (day 8). i COT/CREAT1: cotinine/creatinine sample 1 (day 1); COT/CREAT2: cotinine/creatinine sample 2 (day 8). g

by Clinical Laboratory Services, Department of Pathology, Milton S. Hershey Medical Center, Pennsylvania State University. The measurements were validated by using internal standards. The detection limits for Tx-M and PGI-M are 5 pg/ml and 9 pg/ml, respectively. PGI-M and Tx-M antiserum were ordered from Advanced Magnetics. Statistical Analyses A nonparametric analysis was conducted for all variables because the variability as indicated by the standard deviations was not equal among the three groups as determined by Levene’s test (Milliken & Johnson, 1984) for the following measurements: PGI-M (p = .02), average of day 1 and day 8 salivary cotinine (p = .0001), and urinary cotinine (p = .0001). In contrast, the standard deviations for the Tx-M and urinary creatinines for the three groups were not different (p = .11 and .29, respectively), but these values were also analyzed by the same method for the purpose of

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TABLE 3. Individual nonsmoker data: Nonsmoking household

ETSH (h/day)

NIPS 3 (µg/m )

Tx-Md (pg/mg creatinine)

0 0 0 0.5 0.4 0 0.4 0.9 0.9 0 0.8 0.4 3.3 3.5 1.6 0.3 0 1.3 0 0 0 0 0.6 0.6 0.3 0.3 0

0 0.0 0.0 0.1 0.1 0.1 0.1 0.1 0.2 0.0 0.2 0.0 1.1 1.1 0.1 0.2 0.0 0.3 0 0 0 0.0 0.2 0.2 0.1 0.0 0

87 121 68 82 81 70 77 79 84 55 73 71 135 74 112 115 39 58 115 57 67 76 97 114 111 82 102

0.6 ± 0.9

0.2 ± 0.3

b

Subject number

Gender

Age (yr)

Home

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

M M F M F M F M F M M F M F M F M F M F F F F F M F M

50 15 12 48 45 17 10 43 46 12 36 14 44 43 17 14 47 20 53 42 15 34 48 20 16 44 14

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

a

Mean ± SD

c

85 ± 24

PGI-Me (pg/mg creatinine) 71 82 57 63 74 39 68 41 63 57 60 75 60 67 53 91 66 56 79 72 110 75 83 62 79 64 45 67 ± 15

a

Home: number of smokers in household. ETSH: self-reported ETS exposure duration. c NIPS: nicotine on personal sampler. d PGI-M: 2,3-dinor-6-keto-prostaglandin F1a . b

internal consistency. A Kruskal–Wallis test (Hollander & Wolfe, 1973a) was conducted to evaluate overall differences among the three groups to determine whether the groups represented a single population. This procedure was followed by comparisons for differences in the three possible pairs of groups using a Wilcoxon rank sum test (Hollander & Wolfe, 1973b). RESULTS The 27 nonsmokers from nonsmoking households self-reported a mean ETS exposure duration of 0.6 ± 0.9 h/day, while the 21 nonsmokers


SCOT1 (ng/ml)

f

g

437

h

CREAT2 (mg/dl)

COT/CREAT1i (µg/g creatinine)


SCOT2 (ng/ml)

UCOT1 (ng/ml)

UCOT2 (ng/ml)

CREAT1 (mg/dl)

0 0 0 1 0 0 2 1 1 0 1 0 0 0 1 0 0 1 0 0 0 0 0 1 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 2 0 2 0 0 1 0 0 0 0 0 0 0 0 0

19 5 4 2 4 5 2 57 6 4 9 3 12 9 52 6 0 6 2 0 0 0 3 10 0 5 8

6 6 3 3 7 5 4 14 7 3 68 5 13 23 21 5 0 12 3 2 1 1 7 9 4 6 4

219 157 162 145 100 171 104 185 148 216 86 115 304 117 281 146 50 113 153 66 42 96 61 167 113 105 224

97 162 127 143 123 234 185 257 160 106 258 96 214 182 213 136 87 141 160 119 182 141 170 259 249 99 289

8.7 3.2 2.2 1.4 4.0 2.6 1.9 30.9 4.0 1.6 10.5 2.6 3.8 7.7 19.7 4.1 0.3 5.3 1.3 0.2 0.4 0.2 4.1 5.7 0.1 4.3 3.3

6.1 3.7 2.0 2.1 5.7 1.9 2.1 5.4 4.1 2.8 28.4 5.2 5.9 12.7 9.6 3.7 0.2 8.2 1.6 1.3 0.5 0.7 4.1 3.5 1.4 5.5 1.2

0±1

0±1

9 ± 14

9 ± 13

142 ± 66

170 ± 59

5±7

5±6

e

SCOT1: salivary cotinine sample 1 (day 1); SCOT2: salivary cotinine sample 2 (day 8). UCOT1: urinary cotinine sample 1 (day 1); UCOT2: urinary cotinine sample 2 (day 8). g CREAT1: creatinine sample 1 (day 1); CREAT2: creatinine sample 2 (day 8). h COT/CREAT1: cotinine/creatinine sample 1 (day 1); COT/CREAT2: cotinine/creatinine sample 2 (day 8). f

from smoking households self-reported 2.6 ± 1.1 h/day of ETS exposure. Mothers provided surrogate reports for small children. Mean air nicotine values taken by personal samplers were 0.2 ± 0.3 µg/m3 for the 27 nonsmokers from nonsmoking households and 0.7 ± 1.2 µg/m 3 for the 21 nonsmokers from smoking households (Tables 2 and 3). Urinary cotinine levels were 2.3-fold higher and salivary cotinine levels 4.0-fold higher in nonsmokers living with smokers compared with nonsmokers who did not live with a smoker (Table 4). Urinary concentrations of the prostacyclin metabolite PGI-M and of the thromboxane metabolite Tx-M (Table 4) did not differ between the nonsmokers who lived with at

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TABLE 4. Mean group values for ETS exposure duration, air nicotine concentration, thromboxane and prostacyclin metabolites, salivary cotinine, and urinary cotinine a

Smokers Nonsmokers (smoking household) Nonsmokers (nonsmoking household)

b

PGI-Mc (pg/mg creatinine)

Salivary d cotinine (ng/ml)

Urinary d cotinine (ng/ml)

105 ± 35 74 ± 20

418 ± 231 1.2 ± 1.7

3087 ± 1431 19.6 ± 16.0

67 ± 15

0.3 ± 0.3

8.6 ± 10.8

n 10 21

N.A. 2.6 ± 1.1

N.A. 0.7 ± 1.2

88.1 ± 41.8 79 ± 26

27

0.6 ± 0.9

0.2 ± 0.3

85 ± 24

e

NIPS (µg/m3 )

Tx-Mc (pg/mg creatinine)

ETSH (h/day)

e

a

ETSH: self-reported ETS exposure duration. NIPS: nicotine on personal sampler. c Tx-M (2,3-dinor-thromboxane B2) and PGI-M (2,3-dinor-6-keto-prostaglandin F1a ) metabolites are pooled samples combining urine taken on day 1 and day 8. d Average values for day 1 and day 8 measurements. e Not applicable. b

least one smoker and the nonsmokers who did not live with a smoker (p = .39 for Tx-M; p = .42 for PGI-M). There was a statistically significant higher urinary concentration of PGI-M in the cigarette smokers when compared with either the nonsmokers who lived with at least one smoker (p < .05) or the nonsmokers who did not live with a smoker (p < .05). There was no significant difference in Tx-M excretion in the cigarette smokers relative to either nonsmokers living with a smoker (p = .99) or to nonsmokers in nonsmoking households (p = .53). DISCUSSION In the 21 nonsmokers who live with smokers in this study, the concentration of ETS in the air does not appear to be sufficient to activate their platelets as measured by release of prostacyclin or thromboxane. In contrast, the cigarette smokers had statistically significant increases in urine levels of the prostacyclin metabolite PGI-M, suggesting activated platelet–vessel wall interaction. Elevated levels of prostacyclin excretion have previously been reported in cigarette smokers (Nowak et al., 1987; Lassila et al., 1988; Barrow, 1989). The smokers in this study did not show increased levels of the stable thromboxane metabolite Tx-M. Increased urinary concentrations of prostacyclin metabolite without a concomitant increase in urinary thromboxane metabolite have previously been reported (Vial et al., 1990; Pernow et al., 1991). In this study, the increased prostacyclin excretion seen in the smokers and the absence of an increase in prostacyclin excretion in ETS-exposed nonsmokers could be explained by the large difference in exposure to


439

tobacco smoke. Several researchers have hypothesized that elevated levels of catecholamines play a central role in platelet responses to inhalation of mainstream cigarette smoke. An initial reaction to the inhalation of relatively large amounts of cigarette smoke by smokers compared with ETS-exposed nonsmokers is thought to involve adrenal secretion of the platelet-activating agents epinephrine and norepinephrine into the vascular system (Cryer et al., 1976; Trap-Jensen et al., 1979; Siess et al., 1982). In vitro studies suggest that, unlike thrombin and collagen, platelet activation induced by epinephrine does not result in thromboxane production (Banga et al., 1986). The increase in urinary thromboxane metabolites sometimes reported to be associated with smoking (Rangemark & Wennmalm, 1991; Uedelhoven et al., 1991; Wennmalm et al., 1991; Dotevall et al., 1992) indicates that in vivo, some degree of platelet activation by agents other than epinephrine may occur. When thromboxane release is present, platelets might have first been sensitized to activation by epinephrine and then activated by other agents at local sites of endothelial injury. Therefore, prostacyclin may be released in the absence of thromboxane release as a defensive reaction of the vessel wall in response to epinephrine-induced platelet aggregation associated with cigarette smoking. The increase in prostacyclin excretion in the absence of thromboxane release observed in the cigarette smokers in this study is consistent with this mechanism. Alternatively, prostacyclin may also be released as a defensive response in the presence of thromboxane release. Many epidemiology studies on the relationship between cardiovascular disease and ETS exposure consider “living with a smoker” as a surrogate of ETS exposure (Smith et al., 1992). This assumption is problematic because it fails to take into account the instances in which smokers who live with nonsmokers only smoke outside and the instances where nonsmokers who do not live with a smoker are exposed to ETS in social settings. The small elevations in relative risk for cardiovascular disease sometimes reported to be associated with ETS exposure can be confounded by inadequate classification of subjects into “exposed” and “not exposed” groups. Therefore, the degree of ETS exposure should only be determined through quantitative assessment methods as described in this study (Walker et al., 1989). The interpretation of the results from this study is limited by the modest number of subjects in the study. One of the strengths of this study is the quantitative manner in which ETS exposure was assessed. During the 8day study period, exposure to ETS was determined by personal samplers for nicotine and by questionnaires subdivided by hour for each study day. In addition, urinary and salivary cotinine and creatinine were assessed at the beginning of day 1 and at the end of day 8. Quantitative assessment of exposure will allow the results of this study to be compared with results from future studies. This quantitative assessment of exposure stands in contrast to the qualitative methods used in other studies (Sinzinger &

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