Effects of inhaled carbon monoxide on acute lung injury in mice

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of morbidity and mortality in the intensive care unit, but despite continuing research few effective therapies have been identified. In recent years, inhaled carbon monoxide (CO) has been reported to have cytoprotective effects in several animal models of tissue injury. We therefore evaluated the effects of inhaled CO in three different in vivo mouse models of ALI. Anesthetized C57BL/6 mice were ventilated with oxygen in the presence or absence of CO (500 parts per million) for 1 h before lung injury was induced by lipopolysaccharide (LPS) or oleic acid (OA) administration. Ventilation was then continued with the same gases for a further 2-3 h, with hemodynamic and respiratory parameters monitored throughout. Intratracheal LPS administration induced lung injury with alveolar inflammation (increased lavage fluid neutrophils, total protein, and cytokines). In contrast, intravenous LPS induced a predominantly vascular lung injury, with increased plasma TNF and increased neutrophil activation (surface Mac-1 upregulation and L-selectin shedding) and sequestration within the pulmonary vasculature. Intravenous OA produced deteriorations in lung function, reflected by changes in respiratory mechanics and blood gases and lavage fluid neutrophil accumulation. However, addition of CO to the inspired gas did not produce significant changes in the measured physiological or immunological parameters in the mouse models used in this study. Thus the results do not support the hypothesis that use of inhaled CO is beneficial in the treatment of ALI and ARDS.     

The behaviour of chickens, mice and rats during euthanasia with chloroform, carbon dioxide and ether

Euthanasia of chickens, young and mature rats, and mice was assessed using chloroform, carbon dioxide and ether. Behavioural patterns were recorded to give some indication of the stress involved. Carbon dioxide induced collapse faster (11.2 +/- 0.4 s) than chloroform (18.9 +/- 0.4 s) or ether (greater than 60 s). With regard to the time taken to death, in carbon dioxide mice had the shortest time (48 +/- 10 s) and mature rats had the longest time (135 +/- 10 s). In chloroform, the only difference was the delayed onset of death (127 +/- 10 s) in the chicken. Behavioural patterns were similar for the chicken in carbon dioxide and chloroform, except for wing flapping, even when unconscious, in carbon dioxide. Chloroform is recommended as more aesthetically acceptable for euthanasia of chickens. Carbon dioxide is recommended for the euthanasia of both rats and mice, considering behavioural criteria. Ether is unsuitable as a euthanasia method as it is dangerous, slow acting and an irritant.     

Effects of carbon monoxide and heme oxygenase inhibitors in cerebral vessels of rats and mice

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10(-6) to 10(-4) M). Mouse MCAs, however, dilated by 21 +/- 10% at 10(-4) M CO. Authentic nitric oxide (NO., 10(-10) to 10(-7) M) dilated both rat and mouse MCAs. At 10(-8) M NO., rat vessels dilated by 84 +/- 4%, and at 10(-7) M NO., mouse vessels dilated by 59 +/- 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor delta-aminolevulinic acid (10(-10) to 10(-4) M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 +/- 2% at 3 x 10(-5) M). Importantly, this constriction was unaltered by exogenous CO (10(-4) M) or CO plus 10(-5) M biliverdine (both HO products). In contrast, exogenous CO (10(-4) M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 +/- 1% in response to 10(-5) M CrMP. Magnesium protoporphyrin IX (10(-5) M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries.     

Changes in protein degradation in chickens due to an inflammatory challenge

Tissue-specific changes in protein catabolism were examined in chicks 16 hr following an inflammatory challenge. It was determined that tyrosine was not catabolized or converted to phenylalanine in muscle, thymus, bursa, or spleen. Therefore, rates of tyrosine release from protein were used to estimate rates of protein catabolism in these tissues. Arginine was not catabolized to urea by chick liver; consequently, arginine release from liver protein was used to measure protein catabolism in this tissue. An injection of sheep red blood cells (SRBC) or Escherichia coli did not change rates of protein catabolism in liver or bursa as compared to saline-injected controls. SRBC significantly increased protein catabolism in muscle and spleen by 29 and 15%, respectively. E. coli resulted in significant increases in muscle, spleen, and thymus of 43, 30, and 34%, respectively. These changes in protein catabolism, together with known changes in protein synthesis, suggest that an inflammatory response to SRBC and E. coli result in increased protein accretion in the bursa and liver, and net protein loss from muscle.     

Effects of carbon monoxide releasing molecule-liberated CO on severe acute pancreatitis in rats

Recent studies have suggested that exogenously administered carbon monoxide (CO) is beneficial for resolution of acute inflammation. Severe acute pancreatitis (SAP) is an inflammatory condition which leads to a systemic inflammatory response syndrome (SIRS). In this study, we investigated the role of CO liberated from carbon monoxide releasing molecule-2 (CORM-2) in rats with SAP. SAP was induced by retrograde infusion of 5% sodium taurocholate into the pancreatobiliary duct. Forty Wistar rats were randomly divided into four groups. Sham group was given normal saline after the sham operation. SAP group was treated with normal saline after the induction of SAP. CORM-2 group was injected with CORM-2 (8 mg/kg, i.v.) after the onset of SAP. iCORM-2 group was given iCORM-2 (an inactive compound used as negative control) after SAP induction. All animals were sacrificed at 12 h after the operation. Eighty rats (n = 20 for each group) were monitored for 7 days to observe their survival rates. In another set of experiments, the former three groups received the same treatment as mentioned above. The last group was given ZnPPIX (HO-1 inhibitor) by peritoneal injection at 1 h before the administration of CORM-2 (n = 10 for each group). Serum levels of amylase, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 10 (IL-10) as well as myeloperoxidase (MPO) activity in pancreatic tissue were determined. Histological score, mRNA expression of these cytokines, heme oxygenase-1 (HO-1) expression, HO activity, and nuclear factor κB (NF-κB)-binding activity in the pancreas were also evaluated. Our results showed that compared with SAP group, CORM-2 treatment significantly reduced the serum levels of amylase, TNF-α, and IL-1β, suppressed pancreatic tissue mRNA expression of TNF-α and IL-1β, and decreased MPO activity in the pancreas. In contrast with the pro-inflammatory cytokines, the serum level and pancreatic tissue mRNA expression of IL-10 were markedly increased by the injection of CORM-2. The severity of pancreatic histology and survival rate were also significantly improved by the administration of CORM-2. Treatment with CORM-2 was associated with an increase in HO-1 expression at 12 h after SAP induction. Pretreatment with ZnPPIX had no effect on the production and mRNA expression of these cytokines at 12 h after the development of SAP with the treatment of CORM-2 as compared to CORM-2 group. Furthermore, CORM-2 treatment inhibited the activation of NF-κB in the pancreas. These results indicate that CORM-2-liberated CO exerts protective effects on SAP in rats, and the beneficial effects may be due to the suppression of NF-κB activation and subsequent regulation of NF-κB-dependent expression of cytokines.     

Inhaled carbon monoxide and hyperoxic lung injury in rats

Because carbon monoxide (CO) has been proposed to have anti-inflammatory properties, we sought protective effects of CO in pulmonary O(2) toxicity, which leads rapidly to lung inflammation and respiratory failure. Based on published studies, we hypothesized that CO protects the lung against O(2) by selectively increasing expression of antioxidant enzymes, thereby decreasing oxidative injury and inflammation. Rats exposed to O(2) with or without CO [50-500 parts/million (ppm)] for 60 h were compared for lung wet-to-dry weight ratio (W/D), pleural fluid volume, myeloperoxidase (MPO) activity, histology, expression of heme oxygenase-1 (HO-1), and manganese superoxide dismutase (Mn SOD) proteins. The brains were evaluated for histological evidence of damage from CO. In O(2)-exposed animals, lung W/D increased from 4.8 in normal rats to 6.3; however, only CO at 200 and 500 ppm decreased W/D significantly (to 5.9) during O(2) exposure. Large volumes of pleural fluid accumulated in all rats, with no significant CO treatment effect. Lung MPO values increased after O(2) and were not attenuated by CO treatment. CO did not enhance lung expression of oxidant-responsive proteins Mn SOD and HO-1. Animals receiving O(2) and CO at 200 or 500 ppm showed significant apoptotic cell death in the cortex and hippocampus by immunochemical staining. Thus significant protection by CO against O(2)-induced lung injury could not be confirmed in rats, even at CO concentrations associated with apoptosis in the brain.     

Age-development and inducibility of hepatic glutathione S-transferase activities in mice, rats, rabbits and guinea-pigs

Hepatic glutathione S-transferase (GST) activities towards 1-chloro-3,4-dinitrobenzene (DNCB), 3,4-dichloronitrobenzene (DCNB), sulfobromophthalein (BSP), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), trans-4-phenyl-3-buten-2-one (TPBO) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) were determined in mice, rats, rabbits and guinea-pigs during ageing and after pretreatment with enzyme inducers. Variations were observed in the developmental patterns and in the phenobarbital-, benzo(a)pyrene-, pregnenolone-16 alpha-carbonitrile-, butylated hydroxyanisole-, trans-stilbene oxide-inducibility of hepatic GST activities in the same species towards different substrates. For example, in rats GST activities for EA, DCNB and TPBO increased respectively, 2.3-, 4.8- and 25-fold during age-development, and after treatment with TSO 1.2-, 3.6- and 1.3-fold. Species differences were found in the maturation and in the inducibility of GST activities. For instance, GST activity toward EA at birth is mature in guinea pigs but not in the other species; phenobarbital treatment increased GST activities in mice and rats but not in rabbits and guinea-pigs; treatment with trans-stilbene oxide enhanced GST activity for TPBO 4.5-fold in mice but not at all in rats. It is concluded that hepatic glutathione conjugation exhibits functional heterogeneity which may be due to species dependent variations in the responsiveness of GST isoenzymes to endogenous and exogenous influences.     

Isolectins in Narcissus: complexity, inter- and intraspecies differences and developmental control

Using high resolution ion-exchange chromatography and isoelectric focusing the heterogeneity of the daffodil ( Narcissus sp.) lectin in terms of isolectin composition was analyzed. A survey of about 30 cultivars and species of Narcissus demonstrates (i) that they all contain over 50 different lectin polypeptides and (ii) that there are pronounced inter- and intraspecies differences in the isolectin patterns. Analyses of lectin preparations isolated from different tissues at different developmental stages further indicate that the isolectin composition is tissue specific and developmentally regulated. Finally, affinity chromatography experiments suggest differences in affinity for a mannose-Sepharose 4B column of different isolectins.     

Hydrogen sulfide as an effective and specific novel therapy for acute carbon monoxide poisoning

Hydrogen sulfide (H₂S) has been recognized as a toxic gas and environment pollutant. So, it is seldom regarded as a therapeutic gas. H₂S has been recognized recently as a novel gaseous messenger and serves as an important neuromodulator in the central nervous system. Many researches have been focused on the protective role of H₂S in treatment of several diseases. Like nitric oxide (NO) and carbon monoxide (CO), which are considered as two gaseous transmitters, H₂S has been regarded as the third one. Recent studies provided evidence that H₂S exerted antioxidant and anti-apoptotic effects, which protected neurons, cardiomyocytes, pancreatic β-cells and vascular smooth muscle cells against oxidative stress by scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS). It has been known that multiple factors, including oxidative stress, free radicals and neuronal nitric oxide syntheses as well as abnormal inflammatory responses are involved in the mechanism underlying the brain injury after acute CO poisoning. Studies have shown that free radical scavengers can display neuroprotective properties. Therefore, we hypothesize that H₂S might be an interesting potential strategy for curing acute CO poisoning.     

Teratogenic potential of inhaled carbon monoxide in mice and rabbits.

Pregnant CF-1 mice and New Zealand rabbits were exposed to carbon monoxide at a concentration of 250 ppm for 7 or 24 hours daily during the period of major organogenesis, days 6 through 15 of gestation in mice and 6 through 18 of gestation in rabbits. Carboxyhemoglobin levels in the range of 10--15% were observed in both species (control animals had 0.7% or less). Carbon monoxide was not found to be teratogenic in either species. In mice, a significant increase in the incidence of some minor skeletal variants was observed. One litter in each of the carbon monoxide-exposed groups of mice was completely resorbed; none of the litters of control mice or of control or exposed rabbits were completely resorbed. The fetuses of mice exposed to carbon monoxide for seven hours daily were heavier than control fetuses, and those exposed for 24 hours daily were lighter than control fetuses. The reason for this result is not known.     

Effect of dietary protein, zinc, and carbon monoxide on fetal zinc concentration in mice

Dietary protein and zinc deficiencies known to be detrimental to the developing fetus are common in pregnant women in developing countries. Everyone in modern society is at risk of exposure to carbon monoxide (CO). This study was conducted to observe the effect of dietary protein, zinc, and exposure to CO on the fetal zinc concentrations by factorial experimentation. Pregnant mice of CD-1 strain were maintained on 17% (control) or 9% (deficient) protein diets mixed with deficient, normal (control), or supplemental zinc throughout gestation. The dams in each dietary group were exposed to air (control) or 500 ppm CO in air in environmental chambers from gestation day 8 to gestational day 18. The dams were sacrificed on d 18 and fetal zinc levels were measured by atomic absorption spectrophotometry. Carbon monoxide levels used in this study had no significant effect on fetal zinc concentration in any treatment group. When both dietary protein and zinc levels were normal, the mean fetal zinc concentrations were higher than all other dietary protein/zinc combinations (15.2±6.0 and 14.2±4.1 μg Zn/g of tissue for 0 and 500 ppm CO levels). However, when dietary protein levels were deficient, supplemental zinc increased the fetal zinc concentrations significantly (12.7±3.8 and 13.1±0.3.6 μg Zn/g of tissue, in 0 and 500 ppm CO groups) as compared to zinc-deficient groups (8.7±3.0 and 10.0±3.3 μg Zn/g of tissue in 0 and 500 ppm CO groups). The results of this study may be relevant to populations that experience both marginal zinc and protein diets during gestation.     

Biologic synergisms in rats produced by carbon monoxide and positive ions

Sprague-Dawley weanling rats were chronically exposed to 200 ppm of carbon monoxide alone and later in combination with an atmosphere containing a high ratio of positive ions generated by a tritium source. The period of exposure approximated two months. Although food and water intake of the exposed rats was not statistically different from the control animals inhaling the ambient laboratory atmosphere, the growth rate was significantly depressed in those animals exposed to carbon monoxide. This developmental inhibition was enhanced by the addition of positive ions to the atmosphere. Spontaneous activity of the animals was also decreased upon exposure to CO. In another series of experiments, immature Sprague-Dawley rats were allowed AD LIBITUM food intake and free choice of four solutions, consisting of water, a 5% glucose solution, a 0.5% saccharin solution, and a 5% ethanol solution. Those animals exposed to 200 ppm CO and positive ions exhibited a taste preference for ethanol solution after three weeks of exposure. This preference continued during the entire two-month exposure period. Neurochemical correlates of these responses have been investigated but more data is required to show significant trends. The relevance of reported data to human habitability in the urban environment is presented in theoretical context.

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Zusammenfassung Junge Sprague-Dawley Ratten wurden anfangs einer Konzentration von 200 ppm CO und später einer Mischung von Luft, mit einen hohen Anteil positiver Ionen (Tritium-Generator) ausgesetzt. Versuchsdauer 2 Monate. Die Wachstumsrate der CO-exponierten Tiere war signifikant verlangsamt, obwohl die Futter- und Wasseraufnahme sich nicht signifikant von der der Kontrolltiere unterschied. Diese Entwicklungshemmung war verstärkt nach Zugabe der positiven Ionen in die Atmosphäre. Ebenfalls vermindert war die spontane Laufaktivität bei CO in der Luft. In einem zweiten Experiment wurden junge Ratten gleichen Bedingungen exponiert, wobei sie Futter frei zur Verfügung hatten und ausserdem freie Wahl zwischen 4 Lösungen: Wasser, 5% Glukose, 0.5% Saccharin und 5% Äthanol. Die Tiere, die 200 ppm CO und positiven Ionen ausgesetzt waren, entwickelten nach 3 Wochen einen besonderen Vorzug für die Äthylalkohollösung. Dieses Verhalten blieb während der Versuchsdauer von 2 Monaten erhalten. Die Aufklärung der neurochemischen Beziehungen erfordert weitere Untersuchungen. Die Bedeutung dieser Versuchsergebnisse für den Menschen in Städten wird besprochen.

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Resume On a élevé de jeunes rats de la race Sprague-Dawley dans une atmosphère contenant 200 ppm de CO, puis dans de l'air ayant une teneur élevée en ions positifs (générateur au tritium). L'essai a duré 2 mois. Le taux de croissance des rats exposés au CO fut ralentie de façon significative bien que la consommation en nourriture et en eau n'ait pas différé de façon significative par rapport aux bêtes de contrôle. Ce ralentissement de la croissance a encore été accentué par l'adjonction d'ions dans l'air respiré. La tendance à se déplacer spontanément fut également réduite par la présence de CO. Dans un second essai, on a placé de jeunes rats dans des conditions analogues. Pourtant, en plus de nourriture AD LIBITUM, les animaux pouvaient choisir entre les brevages suivants: eau, solutions de 5% de glucose, 0.5% de saccharine et 5% d'alcool aethylique. Les animaux exposés à de l'air contenant du CO et des ions positifs ont montré, après 3 semaines, une nette prédilection pour la solution d'alcool aethylique. Cette prédilection s'est maintenue durant toute la durée de l'essai (2 mois). Il sera nécessaire de faire de nouveaux essais afin d'expliquer les relations neurochimiques de cause à effet. On discute enfin la portée du résultat de ces essais pour l'homme vivant dans les villes.

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Presented at the 5th Biometeorological Congress, Montreux, Switzerland, 31 August to 6 September 1969. This presentation was made possible through the support of the National Science Foundation.     

Moderate prenatal carbon monoxide exposure produces persistent, and apparently permanent, memory deficits in rats

The effects of moderate (150 +/- 2 ppm) prenatal carbon monoxide (CO) exposure (maternal HbCO concentrations of 15.6 +/- 1.1%) on learning and memory were assessed in young and aged adult rats using a two-way active avoidance paradigm. In experiment 1, the prenatal CO-exposed rats at 120 days of age acquired a conditioned avoidance response equally well as control animals in a 100-trial session. However, following a 24-hr interval the CO-exposed rats failed to demonstrate significant retention of the task as indicated by the absence of significant improvement in performance over the indicated by the absence of significant improvement in performance over the previous day; control subjects did show significant retention. In experiment 2, in which 120-day-old animals received 50 training trials per day until a criterion of ten consecutive avoidance responses was met, the prenatal CO-exposed subjects again acquired the task as well as control animals. When tested for retention 28 days later, a significant memory impairment was again observed in terms of trials required to reattain the avoidance criterion as well as in total percent avoidance responding. In neither experiment did an analysis of initial or average latency to escape the footshock stimulus reveal any significant alterations. These latter results suggest that the observed performance impairment reflected a memory deficit and not a disruption of sensory, motor, or motivational factors. In experiment 3, prenatal CO-exposed rats approximately 1 year of age (300-360 days of age) showed impairment relative to air-exposed controls in both the original learning and retention of the two-way avoidance response. Again, however, there was no evidence for alterations in performance factors per se. Collectively these data indicate that while young adult rats prenatally exposed to 150 ppm CO demonstrate an associative deficit restricted to memory impairment, aged adults similarly exposed during the prenatal period display a more pronounced deficit similar to that recently reported for animals tested as juveniles. The importance of parametric manipulations in uncovering long-term toxicity is also discussed.     

Sex related differences in the response of mice, rats and cats to administration of picrotoxin

Picrotoxin, 2.5 mg/kg, which was subconvulsive in male rats was 92% convulsive in female rats. Four mg/kg of picrotoxin, a dose which did not produce death in the male rats, was 75% lethal in the female rats. Picrotoxin also produced a significantly greater increase in the frequency of the spinal motoneurons discharge in the female than in male rats (444% of control compared to 222% of control). A similar significant difference to the analogous treatment was obtained in the female and male cats (439% of control compared to 368% of control). To counteract the picrotoxin-induced increased frequency of the spinal motoneurons discharge a double dose of diazepam had to be given to females of both species. A sex related difference in the occurrence of convulsions, latency and death following picrotoxin administration was also present in mice. However, mice responded in an opposite direction to rats and cats. Three mg/kg of picrotoxin was 100% convulsive and 27% lethal in male mice, while only 40% convulsive and 0% lethal in female mice. In male mice treated with a 100% lethal dose of picrotoxin, diazepam, 3.0 mg/kg, did not diminish the occurrence of convulsions but reduced the incidence of death to 70%. In equally treated female mice the same dose of diazepam reduced the occurrence of convulsions from 100 to 70% and the incidence of death to 10%. The existence of sex related differences in the response of mice, rats and cats to administration of picrotoxin might have its origin in the dimorphisms of the GABA system in these animal species.