- Email: [email protected]
Postnatal toxicity of carbon monoxide after pre- and postnatal exposure
Toxicology Letters, 1(1977) 147-150 o Elsevier/North-Holland Biomedical Press
147
POSTNATAL TOXICITY OF CARBON MONOXIDE AFTER PRE- AND POSTNATAL EXPOSURE
DAVID J. HOFFMAN and KIRBY I. CAMPBELL Health Effects Research Laboratory, Environmental Research Center, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 (U.S.A.) (Received July 26th, 197’7) (Accepted August 8th, 1977)
SUMMARY
Rats were exposed prenatally through 21 days postnatally to 230 ppm carbon monoxide (CO). Offspring weights at 1 day and 5 days were depressed, and values for carboxyhemoglobin, hematocrit, hemoglobin, and heart weight: body weight ratio were elevated, compared to controls. At 21 days survival was markedly reduced in CO-exposed rats (36%) compared to controls (94%). These results, in view of other studies involving only prenatal or neonatal exposure at higher CO levels, suggest that neonatal and postnatal toxicity of CO may be enhanced by prenatal exposure.
INTRODUCTION
Prenatal exposure of mice and rats to moderate levels of CO during gestation has resulted in few gross teratogenic defects [4,2] and little subsequent effect on postnatal weight gain [ 21. Previous studies in this laboratory have shown that exposure of suckling rats to moderate and higher levels of CO have little effect on survival [ 31. In the present study it was found that when rats were exposed to moderate levels (230 ppm) of CO continuously throughout the prenatal period, parturition, and 21 days postnatally, there was a marked decline in postnatal survival with delayed growth. METHODS
Female random-bred albino rats (Charles River CD stock, Sprague-Dawley descended*) were caged overnight with males of the same stock and a positive *Animals were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Mention herein of commercial names does not necessarily constitute endorsement by the Environmental Protection Agency.
148
sperm smear the following morning was considered as day 0 of gestation. Animals were maintained at controlled temperatures with controlled lighting (12 h of light) and fed commercial chow and water ad libitum. Two exposure atmosphere chambers were used: (1) a purified air (control) chamber, and (2) a test chamber supplied continuously with purified air containing CO* at a concentration of 230 ppm. The CO was supplied in controlled portions from a gas cylinder into the chamber air supply. The ventilation rate for both exposure chambers was 15 vol./h. CO levels were measured automatically by nondispersive infrared spectroscopy on a frequent periodic basis (approximately hourly). Eighteen 2-day pregnant rats were randomly divided into two groups and placed .in either the clean air chamber or the CO chamber. Rats remained in the chambers and respective atmospheres continuously and were permitted to deliver their litters, which also remained there for 21 days postnatally. At 5 days of age, four males from each litter were sacrificed after collecting blood by cardiac puncture. Determinations of hematocrit, hemoglobin by the cyanmethemoglobin method, and carboxyhemoglobin [ 11 were made. Visceral organs from these males were examined and weighed and skeletal preparations were made by clearing with 1% KOH and then staining with Alizarin Red S. Statistical comparisons using the t-test were based on the litter as the experimental unit with the standard deviation representing the variation of litter means. Overall survival rates were compared using the chi square test. RESULTS
AND DISCUSSION
The onset and duration of parturition as well as the percent of live births were similar in both the CO and clean air groups. Neonatal weights at 24 h were significantly lower, by 14%, in the CO group (Table I). By 4 days of age survival had fallen to 72% in the CO group, compared to 96% in the controls, and by 5 days growth (body weight) was impaired by nearly 41% compared to controls. No visceral defects were apparent in males that were sacrificed at 5 days. Heart weight/body weight ratios were significantly greater. When heart weights were compared on an absolute basis (g) this difference was not apparent. Skeletal examination revealed mainly minor variants after CO exposure, including rudimentary 14th ribs, although wavy ribs (5.5% per litter) were also seen. Hematocrit and hemoglobin concentrations were significantly elevated, by 8 and 9% respectively. Carboxyhemoglobin concentrations (24%) were comparable to those found in the mothers (26%). By 21 days survival among the remaining pups in the CO chamber had decreased to less than 36% and the growth rate was significantly lower than that of controls. Exposure of pregnant rats to 1000 ppm CO for up to 9 h or to 300 ppm continuously for up to 6 days during several periods of gestation including the *Carbon
monoxide
was CP grade and supplied
by the Union
Carbide
Corp.
149
TABLE I GROWTH AND SURVIVAL, HEMATOCRIT, HEMOGLOBIN, AND CARBOXYHEMOGLOBIN VALUES IN RATS EXPOSED TO CARBON MONOXIDE a
Litter size Survival; day 4 (W) Survival; day 21 (%) Neonatal weight; 24 h (g) Growth rate; day l-day 21 (g/day) Body weight; B-day-old males (g) Heart weight; 5-day-old males (g/100 g body weight) Spleen weight; 5-day-old males (g/100 g body weight) Hematocrit; 5-day-old males (%) Hemoglobin; 5-day-old males (g%) Carboxyhemoglobin; 5-day-old males (%)
CJean air controls X+ SD
@bon monoxide Xr SD
8.9 96 94 1.3 2.4 11.8
9.2 72h 36b 6.2 1.6 7.0
f 2.8
?- 0.59 + 0.25 * 1.56
0.86 f 0.084 0.70 t 0.10 34.5 + 1.78 10.2 +_0.31 1%
+ 3.1
r 0.69 b f 0.14 b f 1.15 b
1.04 ? 0.093 b 0.74 + 0.23 37.1 +_3.07 c 11.1 k 0.79 b 24%
aValues are expressed as mean f SD. b P < 0.01, t-test. c P < 0.05, t-test.
latter part of gestation did not produce any effect on postnatal weight gain [2]. Previous studies in this laboratory where suckling rats were exposed to 550 ppm CO resulted in only 4% mortality by 21 days of age, with weight gain that was 35% less than that of clean air-exposed controls [3]. The present results, including 64% mortality by 21 days of age and reduction in weight gain by 34% under a considerably lower CO concentration than in published studies, suggest that continuous exposure to moderate levels of CO prenatally, throughout parturition, and for 21 days postnatally is more toxic to the neonate than either prenatal or postnatal exposure alone. Although the exposure conditions of the published studies were not identical to those of the present study, the fact that CO exposure levels were 30 to 430% higher in the published prenatal exposure studies and were almost 230% higher in the published postnatal studies tends to support this assertion. ACKNOWLEDGEMENT
The authors thank Dr. William J. Scott of the Children’s Hospital Research Foundation, Cincinnati, for his helpful manuscript review.
150
REFERENCES 1 I. Davidsohn and D.A. Nelson, Methods used in the study of blood, in I. Davidsohn and J.B. Henry (Eds.), Clinical Diagnosis by Laboratory Methods, W.B. Saunders Co., Philadelphia, 1974. 2 C.T. Grabowski, Postnatal effects on rats of prenatal exposure to carbon monoxide, Teratology;13 (1976) 22A. 3 D.K. Hysell, W. Moore, R. Hinners, M. Malanchuk, R. Miller and J.F. Stara, Inhalation toxicology of automotive emissions as affected by an oxidation exhaust catalyst, Environ. Health Persp., 10 (1975) 57-62. 4 B.A. Schwetz, B.K.J. Leong and R.E. Staples, Teratology studies on inhaled carbon monoxide and imbibed ethanol in laboratory animals, Teratology, 11 (1975) 33A.
147
POSTNATAL TOXICITY OF CARBON MONOXIDE AFTER PRE- AND POSTNATAL EXPOSURE
DAVID J. HOFFMAN and KIRBY I. CAMPBELL Health Effects Research Laboratory, Environmental Research Center, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 (U.S.A.) (Received July 26th, 197’7) (Accepted August 8th, 1977)
SUMMARY
Rats were exposed prenatally through 21 days postnatally to 230 ppm carbon monoxide (CO). Offspring weights at 1 day and 5 days were depressed, and values for carboxyhemoglobin, hematocrit, hemoglobin, and heart weight: body weight ratio were elevated, compared to controls. At 21 days survival was markedly reduced in CO-exposed rats (36%) compared to controls (94%). These results, in view of other studies involving only prenatal or neonatal exposure at higher CO levels, suggest that neonatal and postnatal toxicity of CO may be enhanced by prenatal exposure.
INTRODUCTION
Prenatal exposure of mice and rats to moderate levels of CO during gestation has resulted in few gross teratogenic defects [4,2] and little subsequent effect on postnatal weight gain [ 21. Previous studies in this laboratory have shown that exposure of suckling rats to moderate and higher levels of CO have little effect on survival [ 31. In the present study it was found that when rats were exposed to moderate levels (230 ppm) of CO continuously throughout the prenatal period, parturition, and 21 days postnatally, there was a marked decline in postnatal survival with delayed growth. METHODS
Female random-bred albino rats (Charles River CD stock, Sprague-Dawley descended*) were caged overnight with males of the same stock and a positive *Animals were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Mention herein of commercial names does not necessarily constitute endorsement by the Environmental Protection Agency.
148
sperm smear the following morning was considered as day 0 of gestation. Animals were maintained at controlled temperatures with controlled lighting (12 h of light) and fed commercial chow and water ad libitum. Two exposure atmosphere chambers were used: (1) a purified air (control) chamber, and (2) a test chamber supplied continuously with purified air containing CO* at a concentration of 230 ppm. The CO was supplied in controlled portions from a gas cylinder into the chamber air supply. The ventilation rate for both exposure chambers was 15 vol./h. CO levels were measured automatically by nondispersive infrared spectroscopy on a frequent periodic basis (approximately hourly). Eighteen 2-day pregnant rats were randomly divided into two groups and placed .in either the clean air chamber or the CO chamber. Rats remained in the chambers and respective atmospheres continuously and were permitted to deliver their litters, which also remained there for 21 days postnatally. At 5 days of age, four males from each litter were sacrificed after collecting blood by cardiac puncture. Determinations of hematocrit, hemoglobin by the cyanmethemoglobin method, and carboxyhemoglobin [ 11 were made. Visceral organs from these males were examined and weighed and skeletal preparations were made by clearing with 1% KOH and then staining with Alizarin Red S. Statistical comparisons using the t-test were based on the litter as the experimental unit with the standard deviation representing the variation of litter means. Overall survival rates were compared using the chi square test. RESULTS
AND DISCUSSION
The onset and duration of parturition as well as the percent of live births were similar in both the CO and clean air groups. Neonatal weights at 24 h were significantly lower, by 14%, in the CO group (Table I). By 4 days of age survival had fallen to 72% in the CO group, compared to 96% in the controls, and by 5 days growth (body weight) was impaired by nearly 41% compared to controls. No visceral defects were apparent in males that were sacrificed at 5 days. Heart weight/body weight ratios were significantly greater. When heart weights were compared on an absolute basis (g) this difference was not apparent. Skeletal examination revealed mainly minor variants after CO exposure, including rudimentary 14th ribs, although wavy ribs (5.5% per litter) were also seen. Hematocrit and hemoglobin concentrations were significantly elevated, by 8 and 9% respectively. Carboxyhemoglobin concentrations (24%) were comparable to those found in the mothers (26%). By 21 days survival among the remaining pups in the CO chamber had decreased to less than 36% and the growth rate was significantly lower than that of controls. Exposure of pregnant rats to 1000 ppm CO for up to 9 h or to 300 ppm continuously for up to 6 days during several periods of gestation including the *Carbon
monoxide
was CP grade and supplied
by the Union
Carbide
Corp.
149
TABLE I GROWTH AND SURVIVAL, HEMATOCRIT, HEMOGLOBIN, AND CARBOXYHEMOGLOBIN VALUES IN RATS EXPOSED TO CARBON MONOXIDE a
Litter size Survival; day 4 (W) Survival; day 21 (%) Neonatal weight; 24 h (g) Growth rate; day l-day 21 (g/day) Body weight; B-day-old males (g) Heart weight; 5-day-old males (g/100 g body weight) Spleen weight; 5-day-old males (g/100 g body weight) Hematocrit; 5-day-old males (%) Hemoglobin; 5-day-old males (g%) Carboxyhemoglobin; 5-day-old males (%)
CJean air controls X+ SD
@bon monoxide Xr SD
8.9 96 94 1.3 2.4 11.8
9.2 72h 36b 6.2 1.6 7.0
f 2.8
?- 0.59 + 0.25 * 1.56
0.86 f 0.084 0.70 t 0.10 34.5 + 1.78 10.2 +_0.31 1%
+ 3.1
r 0.69 b f 0.14 b f 1.15 b
1.04 ? 0.093 b 0.74 + 0.23 37.1 +_3.07 c 11.1 k 0.79 b 24%
aValues are expressed as mean f SD. b P < 0.01, t-test. c P < 0.05, t-test.
latter part of gestation did not produce any effect on postnatal weight gain [2]. Previous studies in this laboratory where suckling rats were exposed to 550 ppm CO resulted in only 4% mortality by 21 days of age, with weight gain that was 35% less than that of clean air-exposed controls [3]. The present results, including 64% mortality by 21 days of age and reduction in weight gain by 34% under a considerably lower CO concentration than in published studies, suggest that continuous exposure to moderate levels of CO prenatally, throughout parturition, and for 21 days postnatally is more toxic to the neonate than either prenatal or postnatal exposure alone. Although the exposure conditions of the published studies were not identical to those of the present study, the fact that CO exposure levels were 30 to 430% higher in the published prenatal exposure studies and were almost 230% higher in the published postnatal studies tends to support this assertion. ACKNOWLEDGEMENT
The authors thank Dr. William J. Scott of the Children’s Hospital Research Foundation, Cincinnati, for his helpful manuscript review.
150
REFERENCES 1 I. Davidsohn and D.A. Nelson, Methods used in the study of blood, in I. Davidsohn and J.B. Henry (Eds.), Clinical Diagnosis by Laboratory Methods, W.B. Saunders Co., Philadelphia, 1974. 2 C.T. Grabowski, Postnatal effects on rats of prenatal exposure to carbon monoxide, Teratology;13 (1976) 22A. 3 D.K. Hysell, W. Moore, R. Hinners, M. Malanchuk, R. Miller and J.F. Stara, Inhalation toxicology of automotive emissions as affected by an oxidation exhaust catalyst, Environ. Health Persp., 10 (1975) 57-62. 4 B.A. Schwetz, B.K.J. Leong and R.E. Staples, Teratology studies on inhaled carbon monoxide and imbibed ethanol in laboratory animals, Teratology, 11 (1975) 33A.