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Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20

Nitrogen Dioxide Exposure and Urinary Excretion of Hydroxyproline and Desmosine a

a

a

a

b

John L. Adgate , Holly F. Reid , Robin Morris , Ronald W. Helms , Richard A. Berg , Ping-Chuan a

a

a

a

a

Hu , Pi-Wan Cheng , Ou-Li Wang , Penelope A. Muelenaer , Albert M. Collier & Frederick W. Henderson a

a

University of North Carolina at Chapel Hill , Chapel Hill, North Carolina, USA

b

Department of Biochemistry , University of Medicine and Dentistry of New Jersey , Piscataway, New Jersey, USA Published online: 03 Aug 2010.

To cite this article: John L. Adgate , Holly F. Reid , Robin Morris , Ronald W. Helms , Richard A. Berg , Ping-Chuan Hu , Pi-Wan Cheng , Ou-Li Wang , Penelope A. Muelenaer , Albert M. Collier & Frederick W. Henderson (1992) Nitrogen Dioxide Exposure and Urinary Excretion of Hydroxyproline and Desmosine, Archives of Environmental Health: An International Journal, 47:5, 376-384, DOI: 10.1080/00039896.1992.9938378 To link to this article: http://dx.doi.org/10.1080/00039896.1992.9938378

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Nitrogen Dioxide Exposure and Urinary Excretion

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of Hydroxyproline and Desmosine

JOHN 1. ADGATE HOLLY F. REID ROBIN MORRIS RONALD W. HELMS University of North Carolina at Chapel Hill Chapel Hill, North Carolina RICHARD A. BERG Department of Biochemistry University of Medicine and Dentistry of New Jersey Piscataway, New Jersey PING-CHUAN HU PI-WAN CHENC OU-LI WANG PENELOPE A. MUELENAER ALBERT M. COLLIER FREDERICK W. HENDERSON University of North Carolina at Chapel Hill Chapel Hill, North Carolina ABSTRACT. The relationship between average and peak personal exposure to nitrogen dioxide and urinary excretion of hydroxyproline and desmosine was investigated in a population of preschool children and their mothers. Weekly average personal nitrogen dioxide exposures for subjects who resided in homes with one or more potential nitrogen dioxide source (e.g., a kerosene space heater, gas stove, or tobacco smoke) ranged between 16.3 and 50.6 ppb (30.6 and 95.1 &m3) for children and between 16.9 and 44.1 ppb (12.8 and 82.9 &m3) for mothers. In these individuals, the hydroxyproline-to-creatinine and desmosine-to-creatinine ratios were unrelated to personal nitrogen dioxide exposure-even though continuous monitoring documented home nitrogen dioxide concentration peaks of 100-475 ppb lasting up to 100 h in duration. Significantly higher hydroxyproline-to-creatinine and desmosine-to-creatinine ratios were observed in children, compared with mothers @ < .001 and .003, respectively).

EXPOSURE to high levels of nitrogen dioxide (NOz)is associated with lung injury in experimental animals and humans, but knowledge of the consequences of exposure to low concentrations of NOz is incomplete. Nu376

merous epidemiological investigations'-' have attempted to demonstrate an association between actual exposure to NOz or common sources of NO, and the prevalence of respiratory symptoms or differences in lung Archives of Environmental Health

function, but inconsistent results have been obtained. Therefore, investigators have sought other means to identify potential markers of NO, exposure. Nitrogen dioxide penetrates deep into the lung because of inefficient upper-airway absorption and because of its relatively limited aqueous sol~bility.~,~ In animal models, NO, exposure to 35 ppm (1 ppm NO, 1.88 x 1oJ pg/m3) has been linked to the develop ment of emphysematous changes in the lungg and levels of 30 ppm have been associated with increased urinary excretion of hydroxyproline-an amino acid abundant in collagen.'o Human studies of urinary hydroxyproline excretion and its relationship to NO, exposure have yielded conflicting results. Yanagisawa et al."+'* found that, in a study of 546 Japanese housewives, the urinary hydroxyproline-to-creatinine ratio (H0P:C) was associated statistically with mean personal NO, exposure (47 f 32.5 ppb) and with active and passive smoking. Other researchers have been unable to duplicate these results in other populations. Muelenaer et al." did not identify an increase in urinary H0P:C in young adult males who were exposed experimentally to 0.6 ppm NO, for 4 h/d for 3 consecutive days. Verplanke et al." found that H0P:C was unrelated to NO, exposure, tobacco-smoke exposure, respiratory symptoms, or to pulmonary function in 6- to 9-y-old school children in the Netherlands. Desmosine, a catabolic product of elastin, is another major connective tissue component of the pulmonary interstitiurn. Increased desmosine excretion was demonstrated in infants who required high oxygen concentrations during the first 3 wk of life." Muelanaer et aI.,l3 however, .found no evidence of increased desmosine excretion in the experimental NO, exposure study described above. Our study was designed to examine the relationship between average and peak levels of indoor NO,, and between indoor NO, exposure and urinary excretion of hydroxyproline and desmosine in a group of preschool children and their mothers. Children who resided in homes where the magnitude of NO, exposure varied considerably were selected for study. The design of this study also allowed us to examine the comparability of two field techniques for NO, exposure assessment: (1) personal, passive-diffusion tubes; and (2) fixed-location, continuous chemiluminescent monitoring.

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Materials and methods Study population. Twenty families with children enrolled in the day-care program of the Frank Porter Graham Child Development Center (FPG) in Chapel Hill, North Carolina, were recruited for this study, and informed consent was obtained from each subject prior to enrollment. The study population consisted of 8 African-American and 12 Caucasian families (7 girls, 13 boys; age range, 3 mo-5.5 y). Subjects were considered exposed if their households had at least one of the following NO, sources: kerosene space heater, gas stove, or cigarette smoke. There were 5 subjects in each exposure-source group; 5 control subjects lived in homes where none of these three potential NO, sources exSeptembedoctober1992 [Vd. 47 (No.511

isted. In the exposed groups, it was impossible to completely restrict exposure to a single source of NO2; 2 of the families in the kerosene heater exposure category and 3 in the gas-stove exposure category were also smokers, and 1 of the families in the kerosene heater exposure category also used a gas stove. Descriptions of each family's potential exposure sources and type of central heat source (a potential confounder) in the homes are provided in Table 1. Exposure assessment. Sampling of NO, was performed with fixed-location active chemiluminescent monitors (Model 8101, Bendix Process Instruments Division, Lewisburg, WV) and passivediffusion monitors." The air sampling rate for the chemiluminescent monitors was 0.15 I/min and stripchart recorders (Model H, Speedomax Corporation, North Wales, PA) provided continuous data acquisition. Chemiluminescent monitors were calibrated (ranged of detection: 0.5-500 ppb) with a Bendix portable calibrator (Model 8861-DA) in which nitric oxide was used as a span gas (Matheson Gas Products, Inc., Joliet, IL). Calibration gases were certified by the U.S. Environmental Protection Agency laboratories (Research Triangle Park, NC), and monitors were calibrated at the beginning and end of each week of monitoring. Diffusion monitors were prepared and analyzed at the Harvard School of Public Health.'6,'7 Five percent of the diffusion tubes in each lot were field or laboratory blanks. Data on source-type and duration of use were collected by monitoring technicians at the beginning of each monitored week. The source-use questionnaire consisted of detailed queries regarding daily use of nonvented heatinglcooking sources, and parental and visitor's tobacco smoking habits. Mothers were instructed to refrain from consuming or serving to their children gelatin or gelatintontaining products during the week of monitoring so that potential confounding effects of intake of exogenous collagen would be reduced. Sampling protocol. The homes of subjects were monitored for 1 wk during the heating season (November-April) and for an additional week during the summer (May-October).On the first day of the week monitoting occurred (Sunday), the chemiluminescent monitor was installed, and questionnaire data were collected with regard to heating-unit type and smoking status. In addition, a source-use diary was given to the parents. The teflon inlet of the chemiluminescent monitor was placed so that it sampled air from the family activity room at the approximate height of the child. One passivediffusion tube was attached approximately 5 in. (12.7 cm) from the end of the inlet tube of the chemiluminescent monitor. At the same time, personal passivediffusion tubes, which were to be worn by the child and mother, were opened and attached to their shirts, and the home heating system was checked to confirm fuel source and ventilation. A second chemiluminescent monitor recorded NO, levels in the child's school classroom. Urine samples. Mothers collected their own and their child's first morning urine the first day after each of the two 1-wk monitoring sessions. In the case of nontoilet-trained children, first-void urine samples were 377

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obtained when the children arrived at day care. Samples were stored at - 70 "C for desmosine analysis, and aliquots for hydroxyproline analysis were lyophilized. Analysis of air-monitoring data. Four different measures of NO, exposure were obtained during each week of monitoring for each family that participated; data were obtained from three passive monitors and from one chemiluminescent monitor. Diffusion tube results were reported as parts per billion (ppb). A digitizing tablet (Model 4956, Tektronix Corporation, Beaverton, OR) was used to obtain the area-under-the-curve (AUC) values from the chemiluminescent monitor strip charts. The AUC values were converted to mean ppb NO, per week by dividing total AUC by time monitored. The four response variables used in the measurement of personal and in-home NO, exposure levels were (1) CONH7-7-d in-home chemiluminescent monitoring; (2) PH7D-7-d passive-diffusion tube inhome average from the tube attached to the chemiluminescent monitor inlet; (3)PKID-diffusion tube worn by child for 7 d; and (4) PMOM-diffusion tube worn by the child's mother for 7 d. PKlD and PMOM were individual exposure averages, whereas CONH7 and PH7D were average levels obtained in a subject's primary home environment. Urinary metaboliie quantification. One-miI IiI iter aliquots of lyophilized urine were hydrolyzed in 6N hydrochloric acid, oxidized to pyrole with chloramine T, and

PH7D (ppb Nitrogen Dioxide)

100

i

0.1 1

*

*

10 100 CONH7 (ppb Nitrogen Dioxide) Kerosene Heaters srnokrrr

+

Gas Stove

Controls

Fig. 1. Regression of in-home weekly average NO2levels, measured by static passivediffusion tubes (PH7D), on continuous monitoring weekly averages (CONH7). 378

were extracted with toluene.'8 The resulting hydroxyproline complex was quantified by spectrophotometry. Urinary desmosine content was determined by radioimmunoa~say.'~ Mono-specific rabbit-serum antibodies to a desmosine-albumin conjugate were added to the urine, following hydrolysis. Cellulose acetate filters for the mixture were then measured for radioactivity. Cotinine content (a marker for exposure to tobacco smoke) was measured by radioimmunoassay,20 in which tritiated cotinine and rabbit anticotinine antisera, purchased from Dr. Helen VanVunakis, Brandeis University, was used. Urinar$ creatinine concentrations were measured in the Clinical Chemistry Laboratory of the North Carolina Memorial Hospital. Urinary content of hydroxyproline, desmosine, and cotinine were referenced to urinary creatinine concentration to standardize for individual urine dilution and body mass. Results were expressed as the following ratios: hydroxypro1ine:creatinine (pg/mg [HOP:C]); desm0sine:creatinine (ng/mg [DES:C]); and cotinine:creatinine (ng/mg [COT:C]). Statistical analysis was performed with the SAS software for personal computers.2'

Results Accuracy of passivediffusion tubes. The accuracy of passive-diffusion tubes for the assessment of cumulative NO, exposure in the field was assessed by comparing data obtained with the passive monitor-mounted at the air-sampling inlet of the chemiluminescent monitor (PH7D)-with data obtained via continuous monitoring (CONH7). The regression of PH7D on CONH7 was described by the equation, PH7D = 2.8 + 0.94CONH7 (F = 1 121;df = 1, 38;p = .OOOl),with an r2 of .97 (Fig. 1). Diffusion tubes provided an accurate reflection of NOz exposure; however, estimates of exposure obtained with passive monitors were, on average, 2.8 ppb higher than those obtained with c hemiIuminescent monitors. This difference was statistically significant (Student's t test, p < .013). NO, exposure levels. Summer and winter mean NO, exposure levels for children and their mothers in the four exposure groups defined in Table 1 are listed in Tables 2 and 3. Summer mean in-home NO, exposure levels (CONH7 and PH7D) in the gas stove homes were significantly higher than in control homes; NO, levels in the homes of smokers were not significantly different than in control homes (Table 2). Summer personal exposure levels were significantly different (p = .OO2) from controls for the mothers in the gas stove exposure group, but not for their children = .129). Analysis for elevated NOz levels among individuals who smoked actively or passively versus individuals who did not smoke did not indicate significant differences between the exposed and control groups (data not shown). During the winter months, mean NO, exposure was highest in homes where supplemental heat was obtained from nonvented kerosene space heaters (Fig. 2). Mean NOz levels in the homes with kerosene heaters averaged more than 2-3times the mean levels in homes equipped with gas stoves, and averaged more than 10 Archives of Environmental Health

Table 1.-Potential

NO1Sources in Study Homes

Exposure group and home number

Unvented sources Space heater Gas stove

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Kerosene space heaters: 1 2 3 4 5 Gas stoves: 6 7 8 9 10 Smokers: 11 12 13 14 15 Controls: 16 17

ia

19 20

Table 2.-Mean CatwrVt

1 Fove n

Controls n

Kerosene Kerosene Kerosene Kerosene Kerosene

Propane None None None None

0 1 0

None None None None None

0

61 43 51 50 37

Propane Oil/electric Oillnatural gas Electric Electric

Natural gas Natural gas Natural gas Propane Propane

1 1 0 1 0

57 50 44 42 15

Gas Natural gas Natural gas Propane Propane

None None None None None

None None None None None

1 2 1

62

60 44 24 21

Natural gas Kerosene Natural gas Electric heat pump Natural gas

None None None None None

None None None None None

0

56 45 39 34 12

Electric baseboard Natural gas Electric heat pump Electric heat pump Electric heat pump

CONH7 13.0 f 5.6 0.10 6 6.5 f 2.6 0.110 7 3.8 2.8 7

*

-

Vented heating

1

2 1

0 0 0 0

N 4 Lev& (ppb* f SD) M e a s u d During the Summer for Each Source

Exposure categoty

Smoker P

No. Child's age (mo) smokers

Response variables PH7D PKlD 19.0 f 10.7 0.022 6 7.3 f 4.0 0.361 7 5.0 f 5.1 7

10.5 f 5.6 0.129 6 10.0 f 5.3 0.131 7 6.0 3.8 7

*

PMOM 14.4 f 4.4 0.002 6 7.6 f 2.5 0.304 7 6.2 f 2.3

6

'1 ppb NO2 1.88 mg/m3. tBecause no one used kerosene heaters during the summer, individuals were reassigned according to NO, source(s) present in their homes. *p value for an independent t test, with controls sewing as reference group.

times the mean NO, levels found in control homes. The NOz levels in homes with kerosene heaters were not statistically significantly higher than controls or were not significantly different from the homes with gas stoves; this probably resulted from (a) the widely fluctuating levels found between the homes, (b) intermittent source-use within homes (as ascertained from inspection of continuous monitoring strip charts), and (c) the small sample sizes. Concentrations of NO, in homes equipped with gas stoves were statistically significantly different from concentrations in control homes, as evidenced by measurements of CONH7 and September/oCtober 1992 [Vd. 47 (No. 5)]

PH7D (p = .050 and .101, respectively). Homes in which people smoked, but where there was an a b sence of gas stoves or kerosene heaters, had mean NOz exposure levels that were approximately twice as high as levels observed in control homes; this difference, however, was not statistically significant. Multivariate regression of the three source variables on PKlD and PMOM indicated that during the winter, gas stoves and kerosene heaters accounted for 69% of the mother's and 67% of the child's variability in personal NO, exposure (Table 4). With respect to the children, the kerosene heater variable was significant (p = 379

.0017), and the gas stove variable was only marginally significant (p = .059). In contrast, both kerosene heaters and gas stoves were associated strongly with maternal exposure to NO, fp = .0034 and .006, respectively). Source-use diaries were completed by study subjects during 39 of the 40 weeks during which monitoring occurred. When source and duration of source-use were included in a regression model, in which winter home

200

mb Nitrogen Dioxide

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160

100

60

0

Qas Stove

Kerorene

Smokers

Controls

Fig. 2. Winter weekly average NO, (ppb), grouped by potential sources and measured by continuous monitoring.

Table 3.-Mean Category

NO, levels (ppb f

Exposure category Kerosene

P* Pt n

Gas stove

P n Smoker

P n

I C(rro's I

NO, levels was the dependent variable, the duration of kerosene space heater use was the only predictor variable of significance (t = 5.0, p < .00l), which explained 80°i0 of the in-home variability. The same analysis was conducted for homes with gas stoves, but homes with kerosene heaters were omitted in the analysis; none of the parameters achieved statistical significance (p < .05). During the 40 wk of in-home monitoring, there were only 9 wk during which at least one NO, peak occurred. A "peak" was defined if chemiluminescent monitor strip data indicated that NO, levels exceeded 100 ppb while subjects were in their homes (seven such exposure peaks were recorded during the winter monitoring season). The shortest peak measured in this study was 15 min in duration and was recorded during the summer monitoring season. During the winter, two of the homes with kerosene space heaters had total elapsed times during which NO, levels exceeded 100 ppb of 4 h 10 min and 100 h 10 min, respectively; the latter duration was measured in a mobile home in which a kerosene heater was the sole source of heat. The one home equipped with both a kerosene heater and a gas stove had levels in excess of 100 ppb for more than 199 h. The maximum level observed in this home was 475 ppb, and NO, levels exceeded 200 ppb for more than 56 h. Peak levels that exceeded 100 ppb were observed in four homes equipped with gas stoves; durations of these peak levels ranged from 2 h 50 min to 18 h 15 min. Metabolite excretion. Comparisons of metabolite excretion values for children and mothers were performed with urine samples obtained during the winter (n = 40) because NO, exposure is usually higher during this season (Table 5). Significantly higher H0P:C and DES:C ratios were observed in children, compared with their mothers (p < .001 and .003, respectively). Cotinine-to-creatinine ratios (C0T:C) were used as corroborative evidence to identify exposed individuals.

SD)Measured During the Winter for Each Source

CONH7

Response variables PH7D PKlD

PMOM

70.0 f 81.2 0.118 0.233 5

71.4 f 73.2 0.076 0.215 5

50.6 f 41.6 0.058 0.160 5

44.1 f 41.1 0.077 0.339 5

22.4 f 15.0 0.050 5 12.0 f 12.2 0.350 5

26.5 f 14.2 0.010 5 14.7 f 14.6 0.171 5

23.8 f 17.0 0.056 5 16.9 f 14.4 0.256 4

6.4 f 3.6 5

4.6 f 3.2 5

19.3 f 17.8 0.263 5 16.3 f 13.2 0.294 5 9.5 f 3.5 5

6.8 f 1.0 5

*Independent t test, using controls as the reference group. tlndependent t test, using gas stove as the reference group.

Archives of Environmental Health

Children who were passively exposed to environmental tobacco smoke had C0T:C levels that were significantly different from controls (p = .003). The C0T:C levels of mothers who actively smoked were 100 times greater than the mean C0T:C levels in mothers who did not smoke or who were exposed passively to cigarette smoke. Although children in the nonexposed group had the lowest C0T:C ratios, their mothers had higher mean C0T:C ratios than the mothers in the passively exposed group, which suggested probable exposure of mothers outside of the home. Multiple linear regression analysis was used to test the relationship between HOPC and DES:C and personal NO, exposure. The regression of H0P:C and DES:C on age, PKID, sex, and race indicated that the only parameter that contributed significantly to the predictive value of the model was age (p .04and p .03 for H0P:C and DES:C, respectively Fable 61). In children, H0P:C and DES:C were related inversely to age. The regression of H0P:C and DES:C on PMOM

-

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-

Tabk 4.--MuHwahate Regmssion of PKID and PMOM on NO, source Availability

and race indicated that neither parameter contributed significantly to the predictive value of the model. Overall normalized excretion rates of hydroxyproline and desmosine were not related to personal exposure 0.296 and F = 0.197, respecof children to NO, (F tively) or to personal exposure of their mothers (F 0.0723 and F 0.763, respectively [Figs. 3'and 41). Descriptive statistics for metabolite excretion are presented in Table 7. Mean H0P:C values for the sub jects who experienced NO, peaks at least once during the week monitoring occurred were the same as the mean for all subjects monitored in this study (Tables 5 and 7). Mean H0P:C levels in homes where NO, sources existed (but in which no exposure peaks occurred) were lower than in control homes. Mean DES:C ratios were lower in children who lived in homes in which 1Wppb peak levels occurred, compared with all other homes. Therefore, even in a subset of individuals who were exposed to the highest average and peak NO, levels, mean metabolite excretion rates for children and mothers did not suggest a relationship between NO, exposure and hydroxyproline or desmosine excretion.

-

-

-

Discussion

Parameter Significance @) SE of estimate

PMOM

40.6 0.0017 f 10.9

-

KERO

Parameter Significance @) SE of estimate

35.2 0.0034 f 10.3

10.1 0.208 f 7.8

19.8 0.059 f 9.8

+

Model GAS

+

29.1 0.006 f 9.2

ra -

SMK

.69

7.7 0.526 f 7.6

Extensive use of passive NO, monitors in a wide variety of geographical areas has indicated that mean NO, levels in homes with and without combustion sources range from 4 to 112 ppb.5*'4~22~2' Leaderer et aLZ6also collected passive-diffusion tube and continuousmonitoring data in 13 homes in which all were equip ped with a gas stove and two kerosene heaters. We also used these two monitoring methods to collect our data, and week-long NO, levels, measured via both continuous and integrated methods, were compared from homes with and without combustion sources. In our study, NO, measurements-taken in the same static location in the home, with both continuous active chemiluminescent NO, analyzers and passive-

Table 5.-Winter Season HydmypdinaCreatinine (HOPC), Desm0sine:Creatinine (DEW), and Cotinine:Creatinine(C0T:C) Ratios for ChiMren and Their M o t h

M HOPC W m g ) DESC (ng/mg) COTC (ng/mg) Active smokers @)*

244.4 299.3

C0T:C (ng/mg) Passive smokers @)

103.0 .003 (n 9)$ 15.9 (n 10)

COTC (ndmg) Not exposed at home '

Children (n SD

-

- 19) Range

Mothers (n SD

M

65.3 140.7-434.9 101.9 i%.5-571.7

79.6

50.1 161.0 3748.1 .071 (n 3)t 19.0-224.0 26.6

-

-

19) Range

65.6 10.9-305.2 154.1 63.8-770.6 1806.1 1919-5530 13.3

15.7-51.9

56.8

0-150.4

.564 (n

9.5

0-27.7 (n

-37.86)

-

10)

*t test with "nonexposed" as the reference group. For unbalanced comparisons, separate variance estimates were calculated. tone value not recovered. $One value not recovered.

September/oaober 1992 [Vd. 47 (No. 5)]

H0P:C ratio (uglmg)

1000

Table C.-Results of Multiple Linear Regression of HOPC and DES:C on Personal NOzExposure and Age for Children and Mothers

ChiId ren Model coefficient SE

Mothers Model coefficient SE

p

p

H0P:C

NO1

-0.41 -2.36

0.54 0.46 1.05 0.04

-0.39

-

0.70 0.59 -

NO1

0.9 -3.70

0.89 0.36 1.58 0.03

-0.88

1.66 0.61

Age DES:C Age

-

-

-

lool

m m 0

looor. I* DES:C ratio (ng/mg)

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m

tt

... . .. + . ’. .

100

+

1

10‘ 1

4+ *

+

I

I

I

+

m

+ + +

I I I 1 1 1 1

I

I

I

I I IIII

I

I

1

H0P:C for Children

0 H0P:C for Mothrrr

Fig. 4. Relationship between hydroxypro1ine:creatinine ratio (H0P:C) and personal exposure to NOl for children and mothers.

++

I I IIII

I

I

I

I I IIII

I

I l l

10 100 ppb Nitrogen Dioxide DES:C for Children

f DESC for Mothers

Fig. 3. Relationship between desm0sine:creatinine ratio (DESC) and personal exposure to NO, for children and mothers.

diffusion tubes-revealed highly concordant results. On average, the static-diffusion tubes (PH7D) gave an estimate that was 2.8 ppb more than the chemiluminescent monitors. Paired t tests showed that this difference was significant statistically during the summer (t = 2.283, p = .034), but PH7D and CONH7 values were not significantly different during the winter (t = 0.952, p = .353), during which time average NO, levels were well above the lower limit of accurate detection (600 ppb/h) for passive-diffusion tubes.” In this population, passive-diffusion tubes provided a convenient and reliable measure of average personal NO, exposure, especially during the winter months when exposure is higher. Winter NO, exposure levels averaged 15-50 ppb higher in homes in which gas stoves or nonvented kerosene heaters were used for cooking or heating 382

I

10 100 ppb Nitrogen Dioxide

1

+ ;

.

0

0

I

10

+

-c++

0

than in homes absent these combustion sources. Our measurements of NO, levels in homes equipped with gas stoves were similar to observations made previously with passive-diffusion monitor^.^^,*^ Kerosene heaters proved to be a potent indoor source of NOz; peak and average concentrations of NO, were consistently highest in homes where kerosene heaters were used. Although obtaining detailed questionnaire data regarding source-use information is cumbersome for large-scale epidemiologic studies, our parent-completed, source-use questionnaire data correlated significantly with cumulative NO, exposure during the winter. Levels of NO, in homes where kerosene heaters and gas stoves were used increased proportionately with parent-reported source-use. An additional p r o b tern area for the investigator who is interested in the health effects of NO, is procurement of field information regarding the magnitude of peak exposures to NO,. In our study, continuous monitoring indicating that NO, exposure peaks exceeded 100 ppb during 9 different weeks, of which 7 occurred during the winter monitoring season. Mean NO, values were two to three times higher in winter, and this was the season during which most peak exposures greater than 100 ppb occurred; therefore, it might be expected that if a relationship existed between urinary metabolite excretion and NO, exposure, it would be observed during winter. Despite relatively copious and documented challenges to exposed individuals in this study, no association between urinary H0P:C or DES:C and NO, exposure was found. Mean H0P:C values for all children in the winter (Table Archives of EnvironmentalHealth

Table 7.-Winter Hydmyproline:Creatinine (HOPC), Desmosine:Creatinine (DESC), and Cotinine:Creatlnine (COTX) Ratios for Individuals Exposed to Peak Levels of NO, (> 100 ppb), Individuals Exposed to NOl Sources Absent Peaks, and Control S u b m s Exposure via

Children

-

SD

Range

M

50.1

168.3-319.6

--

244.4 0.487 218.4 280.6

38.0 0.305 26.4 100.4

--

245.5 0.061 325.7 337.8

HOPC crs/mg) Peaks (n 5)

P Sourcest (n 7) Controls (n 7) DES:C (nglmg) Peaks (n 5) P Sources (n 7) Controls (n 7)

-

Mothers

M

metabolite group

42.0 140.7-278.9 99.8 201.4-434.9 35.7

198.4-297.6

141.9 196.5-571.7 80.7 233.9-452.1

129.1 0.305 111.1 275.5

SD

Range

25.3

17.4-92.6

8.0

117.9

10.9-33.9 26.3-305.2

49.1

68.7-192.1

51.5 277.1

63.7-208.2 139.2-770.6

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*Independent sample t tests, with controls as the comparison group. tlndividuals who had NO1 sources in their homes, but no exposure peaks > 100 ppb occurred.

5) and from children who lived in homes where peaks greater than 100 ppb occurred at least once during the winter (Table 7) were fairly equal at 244.4 pg/mg. H0P:C levels in homes where there were NO, sources (but no exposure peaks) were actually lower than in the homes of control subjects (Table 7).Mean DES:C ratios were lower in children who lived in homes where 100-ppb peak levels occurred than in children from all other homes. No urinary metabolite parameters were related to NO, exposure in mothers or children, independent of residence in the seven higher-risk homes. H0P:C values ocglmg) in this study ranged from 140.7 to 434.9, which is a wider range than observed in 4 to 5-y-olds (i.e., 120.6 to 265.5 pglrng2") or 6- to 9-y-olds (i.e., 92.0 to 233.0 pglmg"). King and Star~ h e r 2reported ~ DES:C values for children and normal adult females to be 289 92 nglmg and 143 84 nglmg, respectively. These values are comparable to our DES:C excretion rates of 299 f 102 ng/mg for children and 161 154 ng/mg for mothers. Presumably, the age-related differences in excretion of collagen and elastin metabolites reflect faster bone, skin, and general connective tissue growth, all of which are characteristic of childhood.30The variations in metabolite excretion reported here and elsewhere, coupled with the fact that such a small amount of total body collagen or elastin actually originates from the lung, support the contention that urinary H0P:C and DES:C may not be sensitive indicators of local lung damage from NO, or from other air pollutants, e.g., environmental tobacco smoke. Evidence of lung injury that is the consequence of acute or chronic NO, exposure is most clearly estab lished in animal toxicological research. Dosimetry data in rhesus monkeys indicate that 50-60% of inspired NOl (exposure range, 560-1 710 pg/m3) is retained during quiet re~piration.~' Rodent dosimetry data indicate that acute NO, uptake in pulmonary air spaces is limited by the chemical reaction of NO, with epithelial surface constituents.' Nonetheless, exposure to 2 ppm

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NO, (accompanied by two daily 1-h spikes of 6 ppm) produces pulmonary lesions in the terminal bronchioles and the proximal alveolar regions." Emphysema,Q33 inflammation and increased infectivity,u altered macrophage morphology, and elevated levels of gammaglutamyl transferase in lavage fluids35 have all been associated with NO, exposure in rodents. Increased urinary excretion of hydroxypr~line~,~' and desmosine" have been observed in experimental animals exposed to NO, levels that range from 1 to 30 ppm. Human exposure to NOl concentrations that are suficient to cause significant changes in connective tissue metabolite excretion appears only in accidents.M Evidence of lung injury related to low-level NO, exposure in humans remains limited and contradictory. Whereas one group of investigators"*'2 has observed an association between urinary H0P:C and personal NO, exposure in adult women, other^'^,'' have been unable to substantiate the same relationship in children and adult males. This research found no correlation between personal NO, exposure and urinary H0P:C or DES:C excretion in preschool children or their mothers. Evidence of human dose-response, in which careful documentation of exposure intensity and measurement of connective tissue catabolites in bronchial lavage fluid are used, may be worth examining x)that direct evidence of lung injury from NO, can be obtained.

********** This project has been funded, in part, with federal funds from the United States Consumer Product Safety Commission under Interagency Agreement number CPSC-IAG85-1179and the United States Environmental Protection Agency through Cooperative Agreement CR 81273801 with the Center for Environmental Medicine. The content of this publication does not necessarily reflect the views of the Commission, nor does mention of trade names, commercial products, or organizations imply endorsement or sanction by the Commission. Submitted for publication May 22, 1991; revised; accepted for publication December 23, 1991.

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Requests for reprints should be sent to: John L. Adgate, Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854.

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