Effect of Lung Irradiation on Mice following Paraquat Intoxlcation* Sawang Saenghirunvattana, M.D.; Anawat Sermsuxm, M.D.; Virat Piratchvej, M.D.; Mana Rochanawutanon, M.D.; Sming Kaojarern, M.D.; and Tharathorn Rattananenya, M.D.
To determine the effect of lung irradiation in mice with paraquat poisoning and whether irradiation could prevent lung destruction, we found the the lethal dose for 50 percent survival (LD.) in 50 mice (Swiss strain) was 25 mglkg of body weight. The first control group comprised 15 mice and their lungs were irradiated with 200 rad for seven days. There was no death within six weeks. The lungs at autopsy appeared mildly congested. The control group comprised 15 mice. They were injected with 25 mglkg of paraquat. Sixdied within three days. The experimental group included
ulmonary fibrosis following poisoning with paraP· quat is almost invariably fatal.' Several recent
reports suggested that the pulmonary damage from paraquat might be reversed by whole-lung irradiation.2-4 Failure of radiotherapy to reverse this progressive pulmonary fibrosis was also reported."? Talbot and Barnes" failed to show a definite benefit from radiotherapy; however, three of nine patients survived. Parkins and Fowler" described an elegant study of the effects of paraquat poisoning on lung function in mice. They demonstrated that irradiation given as a single dose three days after the toxin did not reverse paraquat lung injury. Unfortunately, they did not demonstrate histopathologic findings. We attempted to explore this issue further using mice following paraquat intoxication and lung irradiation. MATERIALS AND METHODS
Paraquat hydrochloride was administered in a single intraperitoneal injection to Swiss-strain mice. Initially 10, 20, 30, 40, and 50 mglkg of body weight were given to 50 animals. From this study, the lethal dose for 50 percent survival (LD 50) was 25 mglkg of body weight. A control group of 15 mice (group 1) was irradiated to the lung using 200 radlday for seven days. The mice were fed and observed for six weeks and then killed. Lung tissue was taken for histologic examination. A dose of 25 mg of paraquat per kilogram of body weight was also administered to 15 mice without lung trradtation (group 2). A dose of 25 mg of paraquat per kilogram of body weight was then administered to 14 mice (group 3) and they were irradiated to the lungs on the same day using 200 radlday for seven days. AU *From the Pulmonary unit (Dr. Saenghtrunvattana), Radiotherapy unit (Dr. Piratehvej), Anatomical Patliology unit (Dr. Rochanawutanon), ToxicOlogy Unit (Dr, Kaojarern), and Department of Medicine (Drs. Sermswan and Rattananenya), Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Supported by a research fund from the Faculty of Medicine, Ramathibodi hospital, Mahidol University, Bangkok, Thailand.
14 mice injected with paraquat and with lung irradiation. Eight died within three days (p>O.5). The pathologic findings in the experimental gioup revealed lung congestion and 6brosis that was more pronounced than in the group that received only paraquat. We conclude that radiotherapy is not effective in amelioration of paraquat-induced lung damage in mice. (Cheat 1992; 101:833-35)
I
=
LD. IetbaI close for 50 percent survival
I
animals were fed ad lib and had free access to water. Mice that died spontaneously were examined as soon as possible and tissue was taken for histologic study At death, the thorax was opened and the lungs were immersed in fixative. Tissue blocks, of sizes suitable for light microscopy; were then cut and were processed as 7-um sections, stained with hematoxylin-eosin. Masson trichrome stain of every lung specimen was also prepared for qualitative evaluation of pulmonary 6brosis. Statistical Analysis
To compare the survival rate between group 2 and 3, the Xl test was used to analyze whether there was improvement in survival rates. .
RESULTS
All animals in group 1 remained well for six weeks. Histologic examination of the lungs at six weeks revealed mild congestion and minimal interstitial edema (Fig 1). There was no evidence of pulmonary fibrosis. Group 2 animals remained well for 24 h, but by day 2 they appeared ill and showed respiratory distress. Of the 15 animals receiving 25 mg/kg of body weight of paraquat, six died within three days. After this period, those surviving exhibited signs of improvement with lessening of dyspnea. In group 3, 8 of 14 animals died within 3 days. The survival rate among this group was not statistically significant when compared with group 2 (p = 0.77). Histology
For group 2 animals treated with paraquat alone, at three days the lungs were congested and there was perivascular edema and hemorrhage. This was associated with interstitial infiltration by lymphocytes, plasma, and polymorphonuclear cells. The alveolar spaces contain edematous fluid, erythrocytes, macroCHEST I 101 I 3 I MARCH, 1992
833
"
FIGURE
1. Mice with lun g irradiation.
,'. "
FIGURE 2. Mice with paraquat poisoning; top left : at th ree days and top right at six weeks. Mice with paraquat Intoxicat ion treated with lung Irradia tion at thr ee days (bottom 1'1t) and six wee ks (bottom rigllt ).
phages, polymorphon uclear cells, and fibrin. By six weeks, hemorrhage and ede ma were less marked and the most prominent finding was infiltration of the alveolar walls by mononuclear cells (Fig 2). Mild fibrosis was also noted (Fig 3), For those animals (group 3) tr eated with paraqu at followed by lung irradiation, lungs were markedl y congested. Infiltration of the lungs consisted of ery throcytes, mononuclear ce lls, and polymorphonucl ear cells, This was more marked when compared with group 2. By six weeks, the ede ma decreased but mild fibrosis had also occu rred (F ig 2 and 4).
study. Ou r study rev 'al ed that lung irradiation alone in group 1 did not r 'due ' th six-wee k survival rate of mice. Minimal edema and cellular infiltrates occurred with the dose used . When lung irradiation was given to paraquat-intoxicated mice (group 3), there was no imp rovem ent in the survival rate when compared with group 2 given paraquat alone (p> O.05). We also demon strated that the lungs, among those with irradiation, showed significantl y more ed ern a and cellular infiltration. The
DISC USSIO N
Paraquat, a widel y used herbi cide , is toxic and frequently fatal in man , Th e lung is the principal target organ within which paraquat is concentrated , Its low redox potential with supe roxide as a significant end product renders it lethal to cells and it may well be a radiosensitizer. It has been demonstrated that paraquat and radiatio n are additive . 10 Several recent case reports of radiotherapy given to patients with paraquat intoxication noted clinical improvement.v' However, adverse results have also been published.s" We are therefore conce rned that such unsubstantiated radiation treatm ent might be widely applied in desperately ill patients without adequate 834
FIG URE 3. Th e fibrinou s exudates were shown by Masson-tri ch rom e stai n of the lung of the mice that survived at six wee ks afte r paraquat intoxication without radia tion treatm en t.
Effect 01Lung Irradiation on Mice alter Paraquat Intoxication (Saanghlruvattana ot a/)
ACKNOWLEDGMENT: The authors would like to express their gratitude to Professor Athasit Vejjajiva, Associate Professor Laksana Pochanulcul, Professor Satit Sirisingh, and Professor Henry Wilde for supporting this study. REFERENCES
FIGURE 4. By Masson-trichrome stain, the fibrous collagen materials were demonstrated in the lung of the mice that survived after six weeks of paraquat intoxication plus radiation treatment.
reason that lung irradiation was initiated on the same day as paraquat intoxication in our study was that Parkins and Fowler" had not found that the optimum time for irradiation, showing reduction of lung damage, was prior to day 3 after toxin ingestion. We did not kill our experimental animals at intervals to compare histopathologic features between group 2 and group 3 because we aimed to evaluate improvement of survival rate and maintained the mice for six weeks. We conclude that radiotherapy is not effective in amelioration of paraquat-induced lung damage in mice .
1 Crofton I, Douglas A. Respiratory diseases, 3rd ed. London: Blackwell Scientific Publications, 1981 2 Talbot AB, Barnes MR, Tlng RS. Early radiotherapy in the treatment of paraquat poisoning. Br I Radiol 1988: 61:40.'HJ8 3 Webb DB, Williams MY, Davies BH, lames KW Resolution after radio therapy of severe pulmonary damage due to paraquat poisoning. BMI 1984; 288:1259-60 4 Shirahama M, Sakemi T, Osato S, Sanai T, Rilcitake 0, Wada S. Recovery after radiotherapy from severe interstitial pneumonia due to paraquat poisoning. [pn I Med 1987; 26:385-87 5 Bloodworth LL, Kershaw IB, Sterens PE, Alcock C], Rainford DT. Failure of radiotherapy to reverse progressive pulmonary fibrosis caused by paraquat. Br I Radioll986: 59 :1037-39 6 Savy Fp, Duval G, Her B, Canu P, Fintelz P. Failure of chemotherapy and radiotherapy in pulmonary fibrosis caused by paraquat. Ann Fr Anesth Reanim 1988: 7:159-61 7 Williams MY, Webb DB . Paraquat lung: is there a role for radiotherapy? Hum Toxicoll987; 6:75-81 8 Talbot AB, Barnes MR. Radiotherapy for the treatment of pulmonary complications of paraquat poisoning. Hum Toxicol 1988; 7:325-32 9 Parkins CS, Fowler IF. A cautionary note on the resolution of paraquat lung damage after radiotherapy. Br I Radiol 1985: 58:1137-40 10 Geard CR, Shea CM , Georgsson MA. Paraquat and radiation effects on mouse C.H lar1/1 cells. Radiother Oncol 1984: 10:1407-41
CHEST I 101 I 3 I MARCH. 1992
B35
To determine the effect of lung irradiation in mice with paraquat poisoning and whether irradiation could prevent lung destruction, we found the the lethal dose for 50 percent survival (LD.) in 50 mice (Swiss strain) was 25 mglkg of body weight. The first control group comprised 15 mice and their lungs were irradiated with 200 rad for seven days. There was no death within six weeks. The lungs at autopsy appeared mildly congested. The control group comprised 15 mice. They were injected with 25 mglkg of paraquat. Sixdied within three days. The experimental group included
ulmonary fibrosis following poisoning with paraP· quat is almost invariably fatal.' Several recent
reports suggested that the pulmonary damage from paraquat might be reversed by whole-lung irradiation.2-4 Failure of radiotherapy to reverse this progressive pulmonary fibrosis was also reported."? Talbot and Barnes" failed to show a definite benefit from radiotherapy; however, three of nine patients survived. Parkins and Fowler" described an elegant study of the effects of paraquat poisoning on lung function in mice. They demonstrated that irradiation given as a single dose three days after the toxin did not reverse paraquat lung injury. Unfortunately, they did not demonstrate histopathologic findings. We attempted to explore this issue further using mice following paraquat intoxication and lung irradiation. MATERIALS AND METHODS
Paraquat hydrochloride was administered in a single intraperitoneal injection to Swiss-strain mice. Initially 10, 20, 30, 40, and 50 mglkg of body weight were given to 50 animals. From this study, the lethal dose for 50 percent survival (LD 50) was 25 mglkg of body weight. A control group of 15 mice (group 1) was irradiated to the lung using 200 radlday for seven days. The mice were fed and observed for six weeks and then killed. Lung tissue was taken for histologic examination. A dose of 25 mg of paraquat per kilogram of body weight was also administered to 15 mice without lung trradtation (group 2). A dose of 25 mg of paraquat per kilogram of body weight was then administered to 14 mice (group 3) and they were irradiated to the lungs on the same day using 200 radlday for seven days. AU *From the Pulmonary unit (Dr. Saenghtrunvattana), Radiotherapy unit (Dr. Piratehvej), Anatomical Patliology unit (Dr. Rochanawutanon), ToxicOlogy Unit (Dr, Kaojarern), and Department of Medicine (Drs. Sermswan and Rattananenya), Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Supported by a research fund from the Faculty of Medicine, Ramathibodi hospital, Mahidol University, Bangkok, Thailand.
14 mice injected with paraquat and with lung irradiation. Eight died within three days (p>O.5). The pathologic findings in the experimental gioup revealed lung congestion and 6brosis that was more pronounced than in the group that received only paraquat. We conclude that radiotherapy is not effective in amelioration of paraquat-induced lung damage in mice. (Cheat 1992; 101:833-35)
I
=
LD. IetbaI close for 50 percent survival
I
animals were fed ad lib and had free access to water. Mice that died spontaneously were examined as soon as possible and tissue was taken for histologic study At death, the thorax was opened and the lungs were immersed in fixative. Tissue blocks, of sizes suitable for light microscopy; were then cut and were processed as 7-um sections, stained with hematoxylin-eosin. Masson trichrome stain of every lung specimen was also prepared for qualitative evaluation of pulmonary 6brosis. Statistical Analysis
To compare the survival rate between group 2 and 3, the Xl test was used to analyze whether there was improvement in survival rates. .
RESULTS
All animals in group 1 remained well for six weeks. Histologic examination of the lungs at six weeks revealed mild congestion and minimal interstitial edema (Fig 1). There was no evidence of pulmonary fibrosis. Group 2 animals remained well for 24 h, but by day 2 they appeared ill and showed respiratory distress. Of the 15 animals receiving 25 mg/kg of body weight of paraquat, six died within three days. After this period, those surviving exhibited signs of improvement with lessening of dyspnea. In group 3, 8 of 14 animals died within 3 days. The survival rate among this group was not statistically significant when compared with group 2 (p = 0.77). Histology
For group 2 animals treated with paraquat alone, at three days the lungs were congested and there was perivascular edema and hemorrhage. This was associated with interstitial infiltration by lymphocytes, plasma, and polymorphonuclear cells. The alveolar spaces contain edematous fluid, erythrocytes, macroCHEST I 101 I 3 I MARCH, 1992
833
"
FIGURE
1. Mice with lun g irradiation.
,'. "
FIGURE 2. Mice with paraquat poisoning; top left : at th ree days and top right at six weeks. Mice with paraquat Intoxicat ion treated with lung Irradia tion at thr ee days (bottom 1'1t) and six wee ks (bottom rigllt ).
phages, polymorphon uclear cells, and fibrin. By six weeks, hemorrhage and ede ma were less marked and the most prominent finding was infiltration of the alveolar walls by mononuclear cells (Fig 2). Mild fibrosis was also noted (Fig 3), For those animals (group 3) tr eated with paraqu at followed by lung irradiation, lungs were markedl y congested. Infiltration of the lungs consisted of ery throcytes, mononuclear ce lls, and polymorphonucl ear cells, This was more marked when compared with group 2. By six weeks, the ede ma decreased but mild fibrosis had also occu rred (F ig 2 and 4).
study. Ou r study rev 'al ed that lung irradiation alone in group 1 did not r 'due ' th six-wee k survival rate of mice. Minimal edema and cellular infiltrates occurred with the dose used . When lung irradiation was given to paraquat-intoxicated mice (group 3), there was no imp rovem ent in the survival rate when compared with group 2 given paraquat alone (p> O.05). We also demon strated that the lungs, among those with irradiation, showed significantl y more ed ern a and cellular infiltration. The
DISC USSIO N
Paraquat, a widel y used herbi cide , is toxic and frequently fatal in man , Th e lung is the principal target organ within which paraquat is concentrated , Its low redox potential with supe roxide as a significant end product renders it lethal to cells and it may well be a radiosensitizer. It has been demonstrated that paraquat and radiatio n are additive . 10 Several recent case reports of radiotherapy given to patients with paraquat intoxication noted clinical improvement.v' However, adverse results have also been published.s" We are therefore conce rned that such unsubstantiated radiation treatm ent might be widely applied in desperately ill patients without adequate 834
FIG URE 3. Th e fibrinou s exudates were shown by Masson-tri ch rom e stai n of the lung of the mice that survived at six wee ks afte r paraquat intoxication without radia tion treatm en t.
Effect 01Lung Irradiation on Mice alter Paraquat Intoxication (Saanghlruvattana ot a/)
ACKNOWLEDGMENT: The authors would like to express their gratitude to Professor Athasit Vejjajiva, Associate Professor Laksana Pochanulcul, Professor Satit Sirisingh, and Professor Henry Wilde for supporting this study. REFERENCES
FIGURE 4. By Masson-trichrome stain, the fibrous collagen materials were demonstrated in the lung of the mice that survived after six weeks of paraquat intoxication plus radiation treatment.
reason that lung irradiation was initiated on the same day as paraquat intoxication in our study was that Parkins and Fowler" had not found that the optimum time for irradiation, showing reduction of lung damage, was prior to day 3 after toxin ingestion. We did not kill our experimental animals at intervals to compare histopathologic features between group 2 and group 3 because we aimed to evaluate improvement of survival rate and maintained the mice for six weeks. We conclude that radiotherapy is not effective in amelioration of paraquat-induced lung damage in mice .
1 Crofton I, Douglas A. Respiratory diseases, 3rd ed. London: Blackwell Scientific Publications, 1981 2 Talbot AB, Barnes MR, Tlng RS. Early radiotherapy in the treatment of paraquat poisoning. Br I Radiol 1988: 61:40.'HJ8 3 Webb DB, Williams MY, Davies BH, lames KW Resolution after radio therapy of severe pulmonary damage due to paraquat poisoning. BMI 1984; 288:1259-60 4 Shirahama M, Sakemi T, Osato S, Sanai T, Rilcitake 0, Wada S. Recovery after radiotherapy from severe interstitial pneumonia due to paraquat poisoning. [pn I Med 1987; 26:385-87 5 Bloodworth LL, Kershaw IB, Sterens PE, Alcock C], Rainford DT. Failure of radiotherapy to reverse progressive pulmonary fibrosis caused by paraquat. Br I Radioll986: 59 :1037-39 6 Savy Fp, Duval G, Her B, Canu P, Fintelz P. Failure of chemotherapy and radiotherapy in pulmonary fibrosis caused by paraquat. Ann Fr Anesth Reanim 1988: 7:159-61 7 Williams MY, Webb DB . Paraquat lung: is there a role for radiotherapy? Hum Toxicoll987; 6:75-81 8 Talbot AB, Barnes MR. Radiotherapy for the treatment of pulmonary complications of paraquat poisoning. Hum Toxicol 1988; 7:325-32 9 Parkins CS, Fowler IF. A cautionary note on the resolution of paraquat lung damage after radiotherapy. Br I Radiol 1985: 58:1137-40 10 Geard CR, Shea CM , Georgsson MA. Paraquat and radiation effects on mouse C.H lar1/1 cells. Radiother Oncol 1984: 10:1407-41
CHEST I 101 I 3 I MARCH. 1992
B35
