Delayed Effects of N0 2 Exposure on Alveolar Permeability and Glutathione Peroxidase in Healthy Humans'?

TORBEN R. RASMUSSEN, SC/JREN K. KJAERGAARD, ULRIK TARP, and OLE F. PEDERSEN Introduction

Considerable efforts have been invested in epidemiological and experimental studies to evaluate the effects of human exposure to nitrogen dioxide (N0 2 ) (reviewed by Samet and Utell [1]). Most experimental studies have focused on the establishment of a no-effect level. However, there has been a great diversity in the reported results even within the framework of controlled exposure studies. Some experiments with asthmatic subjects have reported effects of shortterm N02 exposure below 0.5 ppm (2-4), while others have failed to detect anyeffects in asthmatics following exposure as high as 4 ppm (5, 6). Few studies have evaluated a possible delayed toxic response to N0 2 , eventhough this is a characteristic of accidental exposure to high concentrations (7). A delayed response, measured as bronchoalveolar mastocytosis and lymphocytosis, has in a single study been demonstrated following a 20-min exposure to 7 mg N02 / m 3 (3.7 ppm) (8). N02 is a strong oxidizing gas. The dominant theory of the toxic principle is that N02 initiates lipid peroxidation, which subsequently causes cell injury or cell death (9). Animal studies have indicated lipid peroxidation and changes in the antioxidative systems are due to N02 exposure, and some studies have shown that antioxidants like vitamin C may protect against the effects of N02 (10-13). The aim of the present study was to evaluate the effects of exposure to N02 in concentrations just below the current occupational threshold limit value of 3 ppm (measured as time weighted average) (14). We have focused on subjective sensations of mucous membrane irritation, lung function, alveolar permeability, and the antioxidative defense offered by glutathione (GSH) and glutathione peroxidase (GSH-Px). Methods Subjects Fourteen healthy, adult subjects, all nonsmok-

654

SUMMARY Potential toxic effects of prolonged NOz exposure below the current threshold limit value (TLV) were examined in 14 healthy, nonsmoking adults. The subjects were exposed to 2.3 ppm NOz and to clean air for 5 h with a 1-wk Interval between exposures. Physiologic and biochemical measurements were obtained during the exposures and until 24 h after. A 14% decrease In serum glutathione peroxidase activity (GSH-Px) was observed 24 h after the start of the NOz exposure, while Indications of a 22% decrease In alveolar permeability were found 11 h after the start. There were no Indications of mucous membrane Irritation or of decreased lung function during or after NOz exposures. The results support the assumption that a delayed response Is a feature of the human reaction to NOz even below the current TLV of 3 ppm, and they stress the Importance of an extended period of observation In future NOz exposure studies. AM REV RESPIR DIS 1992; 146:654-659

ers, participated in the study (4 females/to males; mean age = 34.4 yr; range: 22-66 yr). All had normal pulmonary function (15)and normal bronchial reactivity (16). None of the subjects had a history of asthma or any regular use of medicine.

ities and the concentration did not exceed 75 ppb with the chamber conditions of the present study.

Design The subjects were exposed in two groups of seven (2 females/5 males) to N0 2and to clean air for 5 h according to a double-blind, crossover design with 1 wk between exposures. Each exposure was preceded by a 2-h cleanair period to provide a baseline level. The effect measurements were repeated until 24 h after the start of the actual exposure. One exposure session is shown schematically in figure 1.

The subjects reported sensations from mucous membranes and of general well-being before, during, and after exposures (figure 1). The sensations were graded on visual analog scales (18). The questions used focused on odor, irritation in eyes, nose, or throat, dyspnea, oppression, and headache.

Exposure Facilities The subjects were exposed in a 74.0 m" climate chamber of stainless steel with to air changes/h, a temperature of 20° C, a relative humidity of 45070, and constant illumination. The air supply passed through charcoal and 99.97% effective particulate filters before N02 was added to the air flowing into the chamber. N02was delivered from a pressurized cylinder with a 1.6% mixture of N0 2in air. The N02level in the chamber was monitored with a Monitor Labs N02 analyzer Model 8840 (San Diego, CA) calibrated with a Monitor Labs Permeator 8840. The N0 2 concentration was raised gradually over a period of 30 min to allow adaptation and avoid possible detection of N02 by odor. Acidic biproducts ofN0 2like nitrous acid (HONO) are inevitably formed in an atmosphere with N0 2 (17), and its presence may complicate the interpretation of N02 effects. The formation of HONO has been measured in our exposure facil-

Measurements Subjective Sensations

Lung Function Lung function parameters were measured before, two times during, and three times after exposures. The following spirometric measures were obtained with a Vitalograph Compacts spirometer (Buckingham, UK): FVC, FEV 10 FEV l/FVC, and forced expiratory flow when 75, 50, and 25% of FVC remained to be expired (MEF,s, MEFso, and MEF 2s). The best curve out of three was selected according to the ATS criteria (19), and the

(Received inoriginalformJuly 3, 1991 andinrevised form March 17, 1992) 1 From the Institute of Environmental and Occupational Medicine, Universityof Aarhus, Aarhus, Denmark. . 2 Supported by Grant No. 1989-06 from The Working Environmental Fund, Denmark. 3 Correspondence and requests for reprints should be addressed to Torben R. Rasmussen, Institute of Environmental and Occupational Medicine, University of Aarhus, Building 180, Universitetsparken, DK-8000 Aarhus C., Denmark.

655

DELAYED EFFECTS OF NO. EXPOSURE

DAY 1:

/Exposure: Air/N021

a

234

567

'u

GSH/Px Sel LF SS

LF

SS

24 25 26 GSH/Px LF SS AP

hours

12 13 14 hours

L.....-_--J'L-'_ _...J

GSH/Px AP

LF

GSH/Px

DAY 2:

8

Sel LF SS AP

LF AP SS

Glutathione I glutathione peroxidase Serum Selenium Lung function Subjective sensations Alveolar permeability

Fig. 1. One exposure session. The subjects were monitored for more than 24 h starting atthe momentthey entered the exposure chamber. The figure shows the time schedule for the different types of measurements.

previously mentioned parameters were taken from this curve. The pneumotachograph was calibrated before each series of measurements by use of a calibrator delivering 8 L by explosivedecompression (20).Peak expiratory flow (pEF) was measured with a Mini-WrightPeakFlow-meter'" (AIRMED, UK). The highest value from three blows was used (21). Total lung capacity (TLC), residual volume (RV), closing volume (CV), and the slope of phase III on the Nitrogen wash-out curve (phase III slope) were measured with Nitrogen wash-out technique using a water-sealed wedge spirometer (22).

Alveolar Permeability The alveolar permeability (AP) was measured as the 99mTechnetium-Diethylenetriamine pentaacetic acid (Tc-DTPA) clearance rate three times following the exposures. Inhaled Tc-DTPAwith a molecular weight of 492 daltons is assumed to pass the alveolar epithelium by a diffusion limited process, and the AP is considered to be a noninvasive assessment of the integrity of the alveolar epithelium (23). It is expressed as "percent cleared per minute" (07o/minute). An aerosol of TcDTPA in 0.9070 saline solution was produced by a Wright nebulizer (15L/min) connected with a closed plastic bag of 25 L placed in an airtight box. The amount of aerosol in the bag could be monitored on a gas-meter by displacement of air from the box (24). The aerosol inhaled from the bag had a count median aerodynamic diameter of 0.78 urn and a mass median aerodynamic diameter (MMAD) of 2.6 urn (24). As soon as the bag was full, the subject would inhale 20 L slowly from the bag in tidal volumes of 2 L from FRC and with a 2-s breathhold before a passiveexhalation. The subject could follow the

inhalation on the gas-meter. At each session the subject would retain 10 to 15 MBq of TcDTPA. Immediately after inhalation, the subject was placed supine on a couch with two scintillation counters over the apices of the lungs. The counters wereplaced here to avoid inclusion of the heart in their fields of view and to keep the absorption of radiation of intervening tissue at a minimum. In that way the activity of the aerosol could be reduced to a minimum. The method was prior to this study compared on seated subjects with simultaneous gamma camera measurements over the back and counters over the apices at the front. The two sets of measurements were highly correlated (pearson's r = 0.96;p < 0.01; slope (SE) = 1.03 (0.15); intercept (SE) = 0.04 (0.17» (unpublished data). The radioactivity was measured over a period of 10 min and the clearance rate calculated as a mono-exponential least square fit to the observed decline in radioactivity (23). Reference values from before exposures were not obtained in order to reduce the radiation dose to a minimum.

Glutathione, Glutathione Peroxidase, and Selenium Total glutathione (reduced + oxidized aSH) concentrations in whole blood and the activity of the selenium dependent glutathione peroxidase (GSH-Px) in whole blood and serum were measured before, immediately after the exposures, and again the following day (figure 1). Blood samples were obtained by venous puncture using dry tubes for serum samples and heparinized tubes for whole blood samples. Preparation of the samples for analysis was started immediately after they were obtained. The samples for the determination of GSH were assayed on the same day im-

mediately after preparation. The samples for assaying GSH-Px and selenium were frozen at -60° C and then analyzed within 4 wk after the collection. All analyses were made in double. aSH was determined by the method of Beutler and associates (25). The coefficient of variation (CV) in whole blood was 7.5070. aSH-px was determined by the method of Paglia and Valentine (26). The decrease in reduced NADPH was followed at 340 nm. T-butyl hydroperoxide was used as substrate. The CV in whole blood was 6.4070 and in serum 4.7070. To assure stable analytical levels, internal standards analyzing GSH-Px were used. The enzyme activity is expressed in katals (kat). One katal corresponds to the conversion of 1 mol ofNADPH per second. The activity in whole blood is expressedas ukat/L, in serum as nkat/g protein. Serum selenium was determined by a fluorometric method as described by Thorling and coworkers (27).

Statistics Statistic analyses were performed with the SPSS/PC + R statistical software package (SPSS Inc., Chicago). Each individual served as his or her own control. The changes from baseline (pre-exposure) level obtained with N0 2 and with clean air were compared using repeated measurements analysis of variance (ANOVA). The pre-exposure values wereused as covariates in the ANOVA if they showed a significant difference between sessions. Recordings of subjective sensations wereanalyzed with Friedman's nonparametric equivalent to a repeated measurements ANOVA. For the alveolar permeability, the response to N0 2 was evaluated by a direct, paired comparison with the values obtained after clean air using Wilcoxon's paired rank test. Correlations were evaluated by Spearman's nonparametric correlation coefficient, rs (28). Unless otherwise stated, the reported p values are two-tailed. Ethics The study was approved by the local ethics committee and performed in accordance with the Helsinki Declaration.

Results All subjects completed all of the measurements.

N02 Exposure The mean N0 2 concentration during exposure was 2.3 ppm (4.4 mg/m") with a maximum range from 2.15 ppm (4.1 mg/m") to 2.64 ppm (5.0 mg/rn"). The background concentration of N0 2 did not exceed 0.03 ppm (0.057 mg/m-).

Subjective Sensations No significant effects were found regarding subjective sensations of mucous membrane irritation in eyes, nose, or airways. All responses to N02 exposure were very small.

656

RASMUSSEN, KJAERGAARD, TARp, AND PEDERSEN TABLE 1 MEASUREMENTS OF LUNG FUNCTION BEFORE, DURING, AND AFTER EXPOSURE Response Attributable to N02 Alone

Parameter Peak Flow, Umin FVC, L FEV1 , L FEV1/FVC, % MEF7li , Usee MEFlio , Usee MEF2li , Usee TLC, L RV, L CV, % ofVC Phase III slope, % N2/L

Type of Exposure

Mean Level before Exposure

Clean air N0 2 Clean air N02 Clean air N02 Clean air N02 Clean air N02 Clean air N02 Clean air N02 Clean air N02 Clean air N02 Clean air N02 Clean air N02

597 601 5.36 5.29 4.10 4.07 76.4 77.2 7.53 7.56 4.30 4.43 1.66 1.64 6.51 6.55 2.20 2.26 14.1 16.5 0.93 1.04

Lung Function Table 1 contains the results of the lung function measurements. The table shows for each parameter their mean pre-exposure values and the response attributable to N02alone. The latter has been calculated by subtracting the mean change from baseline level observed with cleanair exposure from the corresponding change observed with N02. This N02response is negative if the lung function

During N02 Exposure Early, Mean (SE)

After N0 2 Exposure

Late, Mean (SE)

4 (4)

6 H after, Mean (SE)

-2 (5)

-4 (6) 0.12 (0.05)

0.16 (0.05)

P < 0.05

0.07 (0.02)

0.07 (0.05)

0.07 (0.05)

0.15 (0.06)

P < 0.05

-1.0 (0.6)

-0.7 (0.5)

-0.6 (0.7)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1.37 0.80 0.32 0.78 0.08 0.32 0.57 0.48 0.65 1.17 0.88 0.68 1.10 0.22

1.2 0.9 0.6 0.8 0.3 0.2 0.7 0.3 0.6 1.5 1.5 0.0 0.9 0.7

1.5 1.8 0.3 0.5 0.0 0.4 0.7 0.1 0.6 1.3 0.7 1.0 1.1 0.2

Mean (SE)

0.67

0.73

0.73

0.02 (0.22)

0.16 (0.20)

NS

- 0.04 (0.12)

- 0.10 (0.09)

0.04 (0.11)

0.07 (0.11)

NS

0.01 (0.07)

- 0.01 (0.05)

0.02 (0.06)

0.09 (0.09)

NS

- 0.05 (0.13)

-0.15 (0.11)

- 0.21 (0.13)

-0.10 (0.11)

NS

-0.05 (0.11)

- 0.12 (0.09)

-0.15 (0.11)

0.00 (0.10)

NS

1.6 (2.0)

0.3 (1.5)

1.5 (1.3)

-0.05 (0.10)

0.07 (0.11)

is relatively more reduced during N0 2exposure than during clean-air exposure. FVC and FEV 1 showed a very slight improvement during and after N02 exposure, while all other indices of lung function were unaffected.

Alveolar Permeability Table2 shows the Tc-DTPA clearance rate for each subject for each exposure session. The mean values of AP and ~AP

Unitsare %/mlnute. .1.: APNO. - APair.

6H

18 H

a

Air

0.3 0.9 -0.3 -0.3 -0.3 0.2 0.0 -0.2 0.0 -0.2 -0.8 1.0 0.2 -0.5

1.6 0.7 0.5 1.3 0.0 0.3 0.6 0.5 0.6 1.5 0.9 0.7 1.1 0.0

1.4 0.7 0.0 0.7 0.1 0.4 0.4 0.6 0.6 0.8 0.6 0.5 1.0 0.2

-0.2 0.0 -0.5 -0.6 0.1 0.1 -0.2 0.1 0.0 -0.7 -0.3 -0.2 -0.1 0.2

0.74

0.57

-0.16 (0.08)

0.00 (0.13)

NS

0.35 (0.21)

0.03 (0.11)

1H N02

0.2 (0.5)

0.10 (0.15)

Time after End of Exposure

Air

NS

0.13 (0.05)

TABLE 2

Total Mean

3 (7)

Evaluation of N0 2 Effeet

0.15 (0.05)

INDIVIDUAL Te-DTPA CLEARANCE RATES FOR EACH TIME OF MEASUREMENT IN EACH EXPOSURE SESSION + THE TOTAL MEAN OF ALL SIX MEASUREMENTS

Subject

18 H after, Mean (SE)

N02

a

N02

a

1.2 0.3 0.3 0.7 0.0 0.3 0.6 0.8 0.8 1.1 1.2 0.9 1.2 0.0

1.3 0.4 0.2 0.7 0.1 0.3 0.4 0.6 0.7 0.8 0.4 1.0 1.3 0.2

0.1 0.1 -0.1 0.0 0.1 0.0 -0.2 -0.2 -0.1 -0.3 -0.8 0.1 0.1 0.2

0.67

0.60

-0.07 (0.07)

Air

-0.2 (0.7) 0.12 (0.09)

NS NS

at the bottom of table 2 show a delayed decrease in AP 6 h after N02 relative to after clean air. The difference in AP between N02 and clean air at 6 h was significant at a 5070 level, while there was no significant difference between APN02 and APair at 1 h or at 18 h. However, two subjects had total mean AP values below 0.30OJo/min (Subjects 5 and 14), and it could be difficult to detect a further reduction in AP for these subjects. If they were excluded from the analyses, the difference at 6 h became more pronounced, now with a p value of 0.017, which is equivalent to an overall significance level of 0.051 (Bonferroni correction). The exclusion of subjects was based on the 95070 confidence interval for whole lung AP among never-smokers found by Groth and coworkers (29). Figure 2 shows how the difference MP ( = APN02 - APair) at 6 h post-exposure for each individual depended on their total mean AP. At 6 h there was a significant negative correlation [Spearman's r, = -0.63 (P = 0.016)], while there was no significant correlation at 1 h or at 18 h (r, = 0.37 (P = 0.197)and r, = -0.07 (p = 0.802), respectively).

Glutathione and Glutathione Peroxidase The mean (± SD) pre-exposure serum selenium concentration was 88 ± 13 J.1g/L, a level comparable to that found in Danish blood donors (27). A normal selenium level is important for the function of GSH-Px (30). The results of GSH

657

DELAYED EFFECTS OF NO. EXPOSURE

AP(N02) - AP(air), %/min.

0.4,------------------_

0.2

0 0

0

0 ~

0 Fig. 2. The difference between alveolar permeability after NOz and after clean air (APNOz - APair) versus total mean alveolar permeability at 6 h postexposure. Spearman's rs == -0.63; P == 0.Q16.

~

0

-0.2

0

0

0 0

-0.4 0

-0.6

0

o -0.8

L - _ - - - - L - _ - - - - - . J_ _---'-----_------' _ _-'--_-----'-_-----..J

o

0.2

0.4

0.6

0.8

1

1.2

1.4

Total mean alveolar permeability. %/min.

and GSH-Px measurements are shown in table 3. There was a significant difference between the two mean pre-exposure values of whole blood GSH-Px (p = 0.004, paired t test), and this appeared to be related to the subsequent changes from baseline level. To adjust for this, the pre-exposure values were used as covariates in the ANOVA. For comparability this was also done for the two other sets of data reported in table 3. For GSH and GSH-Px in whole blood, no effects of NOz exposure were found, while for GSH-Px in serum, a significant effect of NO z was found (p < 0.05). There was no correlation between changes in alveolar permeability and serum GSH- Px. Discussion No indications of acute or delayed subjective sensations of mucous membrane irritation were found in this controlled

study, even though the subjects were exposed to relatively high concentrations ofNO z (2.3 ppm) for an extended period of time (5 h) compared with the majority of published human studies. There have been diverging results regarding the lower threshold for measurable effects of NO z exposure on lung function among healthy adults (5, 31,32). The majority of studies on healthy subjects have not found any effect of NO z on lung function, but those reporting effects have found a decline in lung function. The apparent difference in FVC and FEV 1 between NO z and clean-air exposures found in the present study is noted from the very start of the exposure and is then unchanged for the whole 24-h observation period. This would be an unlikely response pattern, and we judge that the slight improvement in FVC and FEV 1 in the present study is only apparent and

that it for both parameters may be explained by the small difference in baseline level between the exposure sessions. FVC and FEV 1 are highly correlated for healthy subjects and would be expected to follow the same pattern. The measurements of alveolar permeability to Tc-DfPA (AP) and the evaluation of the antioxidative defense system appeared to be influenced by the NO z exposure. The AP reflects changes in the most peripheral parts of the lungs - the alveolar epithelium (23) and histologic examination of lung specimens from animals exposed to NO z indicates that this is the main target area for NO z (33). Exposures to other oxidizing gases like 1000/0 oxygen (34) or ozone (35) have been shown to result in increased alveolar permeability to Tc-DfPA. Previous assessments of the effects of NO z on alveolar permeability have resulted in diverging conclusions, probably depending on the method used. Ranga and coworkers (36) measured the alveolar permeability to horseradish peroxidase (MW = 40,000 daltons) in guinea pigs after exposures to 5 and 15ppm NO z for 2 and 14 days and found increased transepithelial permeability, which was mainly caused by an increased pinocytotic activity. Only after 14 days with 15 ppm NO z, a transjunctional leak was observed. After a 4-h exposure of rats to 10,20, 30, and 40 ppm NO z , Guth and Mavis (37) found evidence of increased leakage of plasma proteins into the alveoli. In contrast to these observations, three animal studies have consistently shown a decreased alveolar permeability to Tc-DfPA following NO z exposure (38-40). In these experiments, dogs were exposed for 1 h to NO z in concentrations from 5 ppm to 400 ppm. In addition, Man and colleagues (40) found an increased content of albumin in bronchoal-

TABLE 3 MEASUREMENTS OF GLUTATHIONE (WHOLE BLOOD) AND GLUTATHIONE PEROXIDASE ACTIVITY (WHOLE BLOOD AND SERUM) Change from Pre- to Postexposure

Parameter Glutathione, whole blood, mmollL Glutathione peroxidase, whole blood, IJ.katiL Glutathione peroxidase, serum, nkatlg protein

* Preexposure valuesof covariates used.

t Between-subjects

*Within-subjects

SE. SE.

Type of Exposure

Level before Exposure, Mean (SEt)

Mean

(SEt)

Mean

(SEt)

Clean air NOz Clean air NOz Clean air NOz

1.01 (0.03) 0.99 (0.03) 343 (30) 283 (25) 137 (6) 142 (7)

0.03 0.07 -24 11 -4 -6

(0.03) (0.04) (17) (8) (2) (3)

0.04 0.01 -36 19 9 -11

(0.03) (0.03) (19) (14) (5) (5)

o H After

18 H After

p Values of ANOVA-evaluation* of NOz Effect Exposure

Time

Interaction

0.388

0.001

0.802

0.549

0.809

0.424

0.013

0.383

0.023

658

RASMUSSEN, KJAERGAARD, TARp, AND PEDERSEN

veolar lavage (BAL) fluid, which is in agreement with the findings by Guth and Mavis (37). So it appears that there is a discrepancy depending on the method by which AP is assessed. N02 exposure seems to lead to decreased Tc-DTPA clearance rate, which should reflect decreased alveolar permeability to small, electrically neutral, hydrophilic molecules (41). The present study is the first investigation on humans of the effect of N02 on the Tc-DTPA clearance rate. There may be severalexplanations why the alveolar permeability to such molecules would decrease after exposure to N02 • Tc-DTPA is believed to move through intercellulary junctions, and if it is assumed that the Tc-DTPAclearance is by a passive mechanism, the clearance rate may be described by Fick's first law of diffusion: The clearance rate = dQ/dt = -DS de/dx, where D is the diffusion coefficient, S is the area of transfer, de is the concentration gradient, and dx the diffusion distance. A swelling of the epithelial cells (32) could lead to an increase in dx or a decrease in S. A greater amount of fluid in the alveolicould decrease de because of dilution of Tc-DTPA in the alveoli. Horseradish peroxidase, which has an MW nearly 100 times greater than that of Tc-DTPA, was moved across the epithelium mainly by increased pinocytotic activity. This is a totally different mechanism than the passivediffusion by which Tc-DTPA is assumed to move, and this could explain the difference. Furthermore, horseradish peroxidase is biologically active. This is not believed to be the case for Tc-DTPA. Therefore, the transport pattern may be different. The movement of plasma proteins across the epithelium from blood to alveoli goes in the opposite direction and is primarily caused by increased permeability of the endothelium together with leakage through the epithelium. This movement of proteins is also likely to go through different channels than those used by TcDTPA because of much higher molecular weights. The difference in response to 100070 oxygen or ozone and to N02 could be due to differences in their access to the interior of the cells of the epithelium. It has been shown that N02 is mainly removed from the airways by a reactive uptake in which it is converted into N02 radicals (N0 2 -) (42), and is probably unable to pass unchanged through the epithelial lining fluid of the

alveoli (43). This is in contrast to the ab- mans using concentrations of N02 above sorption of oxygen, which is able to cross the TLV(8). The decrease in serum GSHthe epithelial lining fluid and reach the Px may indicate a responseto N02 caused cell. So at least the different alveolar by increased lipid peroxidation, although permeabilities seen in response to N02 the serum measurements can only be a and to 100070 oxygen could be the result reflection of potential changes in the pulof a conversion of N02 before it is able monary cells or lining fluid. Lipid peroxto reach the cells. N02 - is toxic but per- ides are highly toxic, and GSH-Px and haps by other mechanisms and with oth- GSH are parts of the major antioxidant er effects than 100% oxygen. Similar in- system that metabolize or inhibit their formation concerning the uptake of formation. A recent in vitro experiment (43)has shown that reduced GSH inhibits ozone seems not to be available. In the present study, a decrease in al- the transfer of N02 through the pulmoveolar permeability was observed 6 h af- nary epithelial lining fluid. In the prester exposure to N02 relative to after air. ent study, we measured total GSH (reFigure 2 shows a significant negative duced and oxidized), which may be less correlation between ~AP and total mean affected than the reduced GSH alone. AP 6 h post-exposure, and it supports This may partly explain why no change the assumption that subjects with a low was detected in total GSH in whole clearance rate are limited in their ability blood. In vivo studies have investigated to respond to N02 • If the two subjects the effects of N02 on antioxidative dewith the lowest total mean values of AP fense mechanisms using different ex(subjects 5 and 14) were excluded from posure designs [reviewed by Sagai and the analysis, the difference at 6 h became Ichinose (45)]. Chow and coworkers (46) more pronounced, while the other differ- did not find any effect on GSH-Px durences remained nonsignificant. The ex- ing exposure of rats to 2.3 and 6.2 ppm clusion criterion used was based on the N02 for 4 days, although a significant results from Groth and associates (29) in increase was seen in other parts of the which the lower 95% confidence inter- glutathione enzyme system, i.e., glutaval limit for the AP of the whole lung thione reductase (G-Red) and glucose-6was 0.3OJo/min. The fact that 2 out of 14 phosphate dehydrogenase (G-6-PD). subjects (14070) had clearance rates below Menzel and colleagues (47) did not find changes in GSH-Px in mice exposed to the lower 95070 confidence interval for 3.4 ppm N0 2 or guinea pigs exposed to whole lung measurements may be due to considerable regional differences as re- 1 ppm N02 • Sagai and others (48)reportported by Groth and colleagues (29). ed time-dependent fluctuating activity of They found the lowest clearance rates in GSH-Px, G-Red, G-6-PD, 6-phosphothe upper peripheral regions, where our gluconate, disulfide reductase, and sumeasurements wereobtained. It is unlike- peroxide dismutase related to lipid peroxidation in lung homogenates of rats ly that the difference could have been caused by an altered deposition of aeroexposed to 10 ppm N02 for 2 wk. A desol after N02 exposure, because the pressed activity of the enzymes was respirometric assessments of pulmonary corded at day 1 after exposure followed by a significant increase to maximum levfunction did not change. To increase the els from day 5 to day 10. This suggests sensitivity of this method in future studies, the counting time should proba- that induction of the antioxidative probly be increased to 15 min. tective enzyme is a compensatory reEvaluation of the anti oxidative GSHI action against lipid peroxide-induced GSH -Px system showed a decrease in damage. Our findings support the hypothesis GSH-Px enzyme activity in serum 18 h post-exposure, while no alterations were that N02 even in concentrations below the current occupational threshold limit found in whole blood GSH-Px or GSH. A possible explanation could be that the value of 3 ppm (5.7 mg/m") induces a amount of GSH -Px found in the erythrodelayed response in the lungs. The findcytes is much greater than that found in ings stress the importance of an extended period of observation following N02 serum so that there is a greater capacity exposure in future studies. to withstand extra needs. Furthermore, it is likely that the GSH-Px in serum is more readily accessible to the cells of the References lungs (44). 1. Samet JM, Utell MJ. The risk of nitrogen diA delay before response is in agreement oxide: what have we learned from epidemiological with the animal experiments as well as and clinical studies? Toxicol Industrial Health 1990; with the results of a recent study on hu- 6:247-62.

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DELAYED EFFECTS OF NO. EXPOSURE

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