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Prevention of Free Radical Mediated Vascular Permeability Increases in Lung Using Superoxide Dismutase
recognized that nitrofurantoin may result in various forms of lung injury, including progressive pulmonary fibrosis, very little is known regarding the earlier stages of lung injury, a time when the lung lesion may be reversible. To assess nitrofurantoin-induced lung injury at its earliest and potentially most reversible stage, a 2-pronged approach was utilized. First, an in vitro ~r cytotoxicity assay was used to assess the mechanism of direct injury of normal lung parenchymal cells by nitrofurantoin. Second, bronchoalveolar lavage was performed on rats injected with nitrofurantoin as an in vivo index of potential indirect mechanisms of lung injury, ie, the recruitment of activated inHammatory cells to the lower respiratory tract. The data from these studies suggests that nitrofurantoin in vitro has the ability to directly injure normal lung parenchymal cells, probably through oxidant mechanisms, and in vivo is associated with recruitment of neutrophils to the lung parenchyma, ie, a potential indirect mechanism which may further contribute to lung cell injury. The first part of this study investigated the ability of nitrofurantoin to directly injure normal lung parenchymal cells. Nitrofurantoin (I0- 5 to 10- 3 M) was incubated with ~r labeled rat lung explants for 12 bat 3T'C resulting in a release of~r, expressed as a cytotoxic index (CI), of1±1, 7±1, and 43 ± 2, respectively. To assess the potential role of oxidants in the ~ediation of this injury, 2 further experiments were rat lung explants were incuconducted. First, ~!'-labeled bated with nitrofurantoin 10- 3 M plus the following antioxidants: superoxide dismutase 300 U/ml (SOD), catalase 1,100 U/ml (CAT), utocopherol10 JLg/ml (uiDC), ascorbic acid 50 JLg/ml (ASC), dimethyl sulfoxide 1 percent (DMSO) and ethanol 0.1 percent (Etoh). The results are as follows: Nitro+ SOD CAT uiDC ASC DMSO Etoh alone CI 43±2 37±2 27±2 30±2 37±2 37±2 35±3 p value <.05 <.001 <.001 <.05 <.05 <.05 Thus, several antioxidants, especially catalase and utocopherol can signi&cantly reduce the direct injury of normal lung cells by nitrofurantoin. The second experiment to assess the potential role of oxidants in the mediation of nitrofurantoininduced lung cell injury utilized ~r rat lung explants incubated with nitrofurantoin (10- 5 to 10- 3 M) under nol'moxic (21% OJ and hyperoxic (95% OJ conditions. The results are as follows: 10- 5 M 10- 4 M 10- 3M Nitrofurantoin air (CI) 1±2 9± 1 45±2 95'110 1 (CI) 5±1 22±1 62±1 p value >.05 <.001 <.001 Thus, hyperoxia appears to accelerate direct nitrofurantoininduced injury of lung cells, and further suggests that the generation of toxic oxygen radicals by nitrofurantoin may be of critical importance in the mediation of lung cell injury. The second facet of this study employs bronchoalveolar lavage (BAL) in the assessment of rats administered toxic doses ofnitrofurantoin. This in vivo model (Boyd, et al AARD 1979; 120: 93) utilizes nitrofurantoin 300 mglkg as a single dose subcutaneous injection to Sprague-Dawley rats (avel'age weight 200 g) which induces acute respiratory distress with a subsequent LD15 at 48 hours after injection. Surviving rats underwent bronchoalveolar lavage at days 1, 2, 3, and 8 after injection to assess potential alterations in the inflamma528
Days after Macrophages Lymphocr/te8 Neutrophils Eosinophils ('II) ('II) ('II) ('II) Nitrofurantmn
0 1 2 3 8
96±4 65±9 71±8 85±6 95±1
4±2 6±2 3±2 6±2 2±1
0 0 29±10 1±1 25±8 1±1 9±5 1±1 1±1 1±1
tory cell traffic of the lower respiratory tract. Whereas the cell differential of a normal rat consists almost entirely of alveolar macrophages with a few lymphocytes, the lavage cell differential of rats administered nitrofurantoin becomes dramatically altered with an influx of neutrophils into the lower respiratory tract. The marked recruitment of neutrophils to the lungs of these rats is associated temporally with the respiratory distress and subsequent high mortality induced by nitrofurantoin. This suggests that nitrofurantoin-induced lung injury in vivo is associated with an acute inflammatory response to the lung, ie, recruitment of neutrophils, which may potentially exacerbate the underlying lung injury. Thus, this model of nitrofurantoin-induced injury suggests that the drug may result in acute lung injury by both direct and indirect mechanisms. The direct injury is likely related to nitrofurantoin's ability to generate toxic oxygen radicals within lung parenchymal cells. This injury in vitro can be significantly reduced by several antioxidants as well as markedly exacerbated in the presence of hyperoxia. A potential indirect mechanism of injury by nitrofurantoin is demonstrated by the recruitment of neutrophils to the lung parenchyma, suggesting that neutrophil-mediated injury may also represent an important mechanism by which ·nitrofurantoin may injure the lung. This study presents preliminary evidence to indicate that nitrofurantoin, perhaps like other drugs toxic to the lung parenchyma, may employ more than one mechanism to effect this tissue injury.
Prevention of Free Radical Mediated Vascular Permeability Increases In Lung Using Superoxlde Dlsmutase* J C. Harker, M.D.; D. J Martin, M.D.; G. RuUU, M.D.;
J McCord, M.D.; and A
E. TIJfllor, M.D.
Infusion of a-naphthyl thiourea (ANTU) in dogs produces a consistent increase in pulmonary microvascular permeability accompanied by pulmonary edema. 1 Since a significant inflammatory response was observed in histologic sections of ANTU damaged lungs, the free radical scavenger, superoxide dismutase (SOD), was administered with ANTU to minimize the action of superoxides on the vascular endothelium. In other experiments, catalase was infused to reduce peroxides generated by the ANTU insult. Mongrel dogs were anesthetized, heparinized, the chest *From the.Department of Physiology, College of Medicine University of South Alabama, Mobile. ' ReJnint reqii!Jst&: Dr. Harker, D~rlment of Physiology, University of South AltJbGmtJ College of Medicine, Mobile 36688 Lung Defenle, Injury and Repair
'Thble 1-E./fec:fa rf Supero:dde Diltnutaae on Vaac:ular Preaaura, Lvmph ftJrameten and Lung Water FollotDing ANTU
J.
N=10 Control Hesponders (N = 5) Nonresp (N = 5) ANTU+SOD Responders Nonresp ANTU +SOD+ t LAP Responders Nonresp
(tAlmin)
CJC,
PAP (cmH10)
LAP (cmH10)
WID
24.5 24.6
0.73 0.67
23.8 27.6
5.4 5.4
3.97 4.04
39.6 137.6
0.65 0.68
33.4 44.0
4.6 5.0
4.13 5.73
183.2 319
0.52 0.62
44.0 50.0
19.8 20.0
4.57 7.14
Table !-Effect cf CattJa.e Or& Lung Lr/mpla ftJrameten.following ANW lnjurv N=3 Control ANTU+
Catalase
J.
(jL}Imin)
c,
Clearance (t.Ll/min)
SAP (mm Hg)
PAP (cmH10)
LAP (cmH10)
P. (cmH10)
(Umin)
52.3
.63
10.6
127.3
16.2
5
11.3
.73
34.2
47.6
.72
48.0
103.3
27.7
2.7
12.0
.42
31.6
46.5
.67
97.1
45.0
31.7
12.7
20.3
~)
18.0
33.7
66.2
Increased
LAP
178
co
C/C,
CL ~)
opened and a small prenodallung lymphatic cannulated. A left atrial balloon catheter was sutured into place, and catheters were also placed into the pulmonary and femoral arteries. The variables measured were: left atrial pressure (LAP), pulmonary arterial pressure (PAP), thermal dilution cardiac output (CO), lymph ftow 0.), total protein concentration in plasma (C.,) and lymph (CJ, and post-mortem extravascular lung water (Q.). Once all variables had attained a steady-state, 5 mg/kg ANTU dissolved in propylene glycol were infused intravenously and all parameters monitored fur 3 hr. Then, left atrial pressure was elevated fur an additional 2 hr. The osmotic reftection coefficient fur total protein was evaluated using: ad= 1- (CJC,) minimal, where (CJC,) minimal is the lowest steady-state lymph to plasma concentration ratio attained at elevated LAP. In ANTU-treated dogs, total protein ad is decreased to 0.40 from 0.65 observed fur uninjured lungs. 1 After ANTU, Q. typically increases from a control value of3.80±0.16 rfg blood free dry wt (BFDW) to 5.87±0.25 rfg BFDW at 3 hr and 7.55±0.55 rfg BFDW after the 2 hr period of increased LAP.' When 2 mg/kg SOD was infused with ANTU in 10 dogs, a variable response was observed. In approximately half (n = 5) the dogs (responders), the increased vascular permeability and pulmonary edema were largely prevented (Thble 1). In others (n =5) there was no apparent beneficial effect (nonresponders). In the responder group, lymph protein clelll'ance ([CJC,)xJ.) increased by 5.3 times control with increased LAP compared to ll.8 times control fur nonresponders, and a lower CL/C, ratio was attained in the resj,onder group. Q. increased only 15% after increased LAP in re-
sponders, compared to 77% fur nonresponders. No relationship of this beneficial response was observed to blood levels of SOD, or arterial Po., Pco1 or pH. The results of similar experiments (n =3) using 8 mg/kg of catalase with ANTU are summarized in 'Illble 2. There was no significant decrease in the minimal CJC, ratio or increased lymph protein clearance after catalase, compared to ANTU alone. The effectiveness of SOD in preventing endothelial damage in 50% of the animals clearly indicates that such damage was mediated by free radicals. In addition, the ineffectiveness of catalase implies that hydroxyl radicals or hydrogen peroxide from tissue sources are not the primary mechanism of injury. Although leukocyte counts were not systematically measured in these animals, the variability may be attributed to differences in the leukocyte response. The beneficial response of SOD could be derived from its effect on plasma leukotaxic factors activated by superoxide but not peroxides, 3 or by interrupting the release of other humoral intermediates involved in lung damage. REFERENCES 1 Rutili G, Kvietys P, M~ D, Parker JC, 1ilylor AE. Increased pulmonary microvascular permeability induced by a-naphthyl thiourea. J Appl Physiol1982; 52:1316-23 2 Parker JC, Parker RE, Granger ON, lilylor AE. Vascular pel'meability and transvascular fluid and protein. Circ Res 1981; 48:545-61 3 McCord JM, Wong K, Stokes SH, Petrone WF, English D. Superoxide and inflammation: a mechanism ror the anti-inflammatory activity of superoxide dismutase. Acta Physiol Scand 1980; (Suppl) 492:25-30
CHEST I 83 I 5 I May, 1983 I Supplement
538
Days after Macrophages Lymphocr/te8 Neutrophils Eosinophils ('II) ('II) ('II) ('II) Nitrofurantmn
0 1 2 3 8
96±4 65±9 71±8 85±6 95±1
4±2 6±2 3±2 6±2 2±1
0 0 29±10 1±1 25±8 1±1 9±5 1±1 1±1 1±1
tory cell traffic of the lower respiratory tract. Whereas the cell differential of a normal rat consists almost entirely of alveolar macrophages with a few lymphocytes, the lavage cell differential of rats administered nitrofurantoin becomes dramatically altered with an influx of neutrophils into the lower respiratory tract. The marked recruitment of neutrophils to the lungs of these rats is associated temporally with the respiratory distress and subsequent high mortality induced by nitrofurantoin. This suggests that nitrofurantoin-induced lung injury in vivo is associated with an acute inflammatory response to the lung, ie, recruitment of neutrophils, which may potentially exacerbate the underlying lung injury. Thus, this model of nitrofurantoin-induced injury suggests that the drug may result in acute lung injury by both direct and indirect mechanisms. The direct injury is likely related to nitrofurantoin's ability to generate toxic oxygen radicals within lung parenchymal cells. This injury in vitro can be significantly reduced by several antioxidants as well as markedly exacerbated in the presence of hyperoxia. A potential indirect mechanism of injury by nitrofurantoin is demonstrated by the recruitment of neutrophils to the lung parenchyma, suggesting that neutrophil-mediated injury may also represent an important mechanism by which ·nitrofurantoin may injure the lung. This study presents preliminary evidence to indicate that nitrofurantoin, perhaps like other drugs toxic to the lung parenchyma, may employ more than one mechanism to effect this tissue injury.
Prevention of Free Radical Mediated Vascular Permeability Increases In Lung Using Superoxlde Dlsmutase* J C. Harker, M.D.; D. J Martin, M.D.; G. RuUU, M.D.;
J McCord, M.D.; and A
E. TIJfllor, M.D.
Infusion of a-naphthyl thiourea (ANTU) in dogs produces a consistent increase in pulmonary microvascular permeability accompanied by pulmonary edema. 1 Since a significant inflammatory response was observed in histologic sections of ANTU damaged lungs, the free radical scavenger, superoxide dismutase (SOD), was administered with ANTU to minimize the action of superoxides on the vascular endothelium. In other experiments, catalase was infused to reduce peroxides generated by the ANTU insult. Mongrel dogs were anesthetized, heparinized, the chest *From the.Department of Physiology, College of Medicine University of South Alabama, Mobile. ' ReJnint reqii!Jst&: Dr. Harker, D~rlment of Physiology, University of South AltJbGmtJ College of Medicine, Mobile 36688 Lung Defenle, Injury and Repair
'Thble 1-E./fec:fa rf Supero:dde Diltnutaae on Vaac:ular Preaaura, Lvmph ftJrameten and Lung Water FollotDing ANTU
J.
N=10 Control Hesponders (N = 5) Nonresp (N = 5) ANTU+SOD Responders Nonresp ANTU +SOD+ t LAP Responders Nonresp
(tAlmin)
CJC,
PAP (cmH10)
LAP (cmH10)
WID
24.5 24.6
0.73 0.67
23.8 27.6
5.4 5.4
3.97 4.04
39.6 137.6
0.65 0.68
33.4 44.0
4.6 5.0
4.13 5.73
183.2 319
0.52 0.62
44.0 50.0
19.8 20.0
4.57 7.14
Table !-Effect cf CattJa.e Or& Lung Lr/mpla ftJrameten.following ANW lnjurv N=3 Control ANTU+
Catalase
J.
(jL}Imin)
c,
Clearance (t.Ll/min)
SAP (mm Hg)
PAP (cmH10)
LAP (cmH10)
P. (cmH10)
(Umin)
52.3
.63
10.6
127.3
16.2
5
11.3
.73
34.2
47.6
.72
48.0
103.3
27.7
2.7
12.0
.42
31.6
46.5
.67
97.1
45.0
31.7
12.7
20.3
~)
18.0
33.7
66.2
Increased
LAP
178
co
C/C,
CL ~)
opened and a small prenodallung lymphatic cannulated. A left atrial balloon catheter was sutured into place, and catheters were also placed into the pulmonary and femoral arteries. The variables measured were: left atrial pressure (LAP), pulmonary arterial pressure (PAP), thermal dilution cardiac output (CO), lymph ftow 0.), total protein concentration in plasma (C.,) and lymph (CJ, and post-mortem extravascular lung water (Q.). Once all variables had attained a steady-state, 5 mg/kg ANTU dissolved in propylene glycol were infused intravenously and all parameters monitored fur 3 hr. Then, left atrial pressure was elevated fur an additional 2 hr. The osmotic reftection coefficient fur total protein was evaluated using: ad= 1- (CJC,) minimal, where (CJC,) minimal is the lowest steady-state lymph to plasma concentration ratio attained at elevated LAP. In ANTU-treated dogs, total protein ad is decreased to 0.40 from 0.65 observed fur uninjured lungs. 1 After ANTU, Q. typically increases from a control value of3.80±0.16 rfg blood free dry wt (BFDW) to 5.87±0.25 rfg BFDW at 3 hr and 7.55±0.55 rfg BFDW after the 2 hr period of increased LAP.' When 2 mg/kg SOD was infused with ANTU in 10 dogs, a variable response was observed. In approximately half (n = 5) the dogs (responders), the increased vascular permeability and pulmonary edema were largely prevented (Thble 1). In others (n =5) there was no apparent beneficial effect (nonresponders). In the responder group, lymph protein clelll'ance ([CJC,)xJ.) increased by 5.3 times control with increased LAP compared to ll.8 times control fur nonresponders, and a lower CL/C, ratio was attained in the resj,onder group. Q. increased only 15% after increased LAP in re-
sponders, compared to 77% fur nonresponders. No relationship of this beneficial response was observed to blood levels of SOD, or arterial Po., Pco1 or pH. The results of similar experiments (n =3) using 8 mg/kg of catalase with ANTU are summarized in 'Illble 2. There was no significant decrease in the minimal CJC, ratio or increased lymph protein clearance after catalase, compared to ANTU alone. The effectiveness of SOD in preventing endothelial damage in 50% of the animals clearly indicates that such damage was mediated by free radicals. In addition, the ineffectiveness of catalase implies that hydroxyl radicals or hydrogen peroxide from tissue sources are not the primary mechanism of injury. Although leukocyte counts were not systematically measured in these animals, the variability may be attributed to differences in the leukocyte response. The beneficial response of SOD could be derived from its effect on plasma leukotaxic factors activated by superoxide but not peroxides, 3 or by interrupting the release of other humoral intermediates involved in lung damage. REFERENCES 1 Rutili G, Kvietys P, M~ D, Parker JC, 1ilylor AE. Increased pulmonary microvascular permeability induced by a-naphthyl thiourea. J Appl Physiol1982; 52:1316-23 2 Parker JC, Parker RE, Granger ON, lilylor AE. Vascular pel'meability and transvascular fluid and protein. Circ Res 1981; 48:545-61 3 McCord JM, Wong K, Stokes SH, Petrone WF, English D. Superoxide and inflammation: a mechanism ror the anti-inflammatory activity of superoxide dismutase. Acta Physiol Scand 1980; (Suppl) 492:25-30
CHEST I 83 I 5 I May, 1983 I Supplement
538