Prenatal cadmium exposure alters postnatal immune cell development and function

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl₂ (10 ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7 weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7 weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2 weeks of age. At 7 weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4⁺FoxP3⁺CD25⁺ (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8⁺CD223⁺ T cells were markedly decreased in the spleens in all offspring at 7 weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.     

密码子优化的HPV6b L1 DNA诱导BALB/c小鼠产生增强的免疫应答

目的 探讨真核细胞偏好密码子优化对人乳头瘤病毒(human papillomavirus,HPV)6b型L1 DNA诱导BALB/c小鼠特异性体液和细胞免疫应答的增强效应.方法 利用PCR从尖锐湿疣组织中扩增HPV6b L1基因,将其克隆至真核表达质粒pcDNA3.1(+),构建野生型pcDNA3.1(+)/HPV6bL1(wt)并进行鉴定;同时,对HPV6b L1基因进行真核细胞偏好密码子优化,进一步通过重叠PCR的方法获得全长基因,构建密码子优化的重组质粒pcDNA3.1(+)/HPV6bL1(mod.)并进行鉴定.将6～8周龄雌性BALB/c小鼠分成4组,分别肌肉注射3次pcDNA3.1(+)/HPV6bL1(mod.)、pcDNA3.1(+)/HPV6bL1(wt)、pcDNA3.1(+)载体和PBS,间隔2周,每次剂量为150μg/鼠.分别于免疫前和免疫后每隔2周,收集血清,用间接ELISA检测血清igG抗体;并于第8周取小鼠脾细胞,采用乳酸脱氢酶释放法,按效应细胞与靶细胞之比(E:T)为5:1、10:1、20:1进行免疫小鼠特异性CTL杀伤活性检测.结果 成功构建了密码子优化的HPV6b L1重组质粒.小鼠免疫后血清特异性IgG抗体水平随着免疫时间和次数的增加而升高.pcDNA3.1(+)/HPV6bL1(mod.)组血清IgG抗体在第8周达到高峰,与pcDNA3.1(+)/HPV6bL1(wt)、载体和PBS对照组相比差异均有统计学意义(t=18.138、t=29.140、t=30.840,P值均＜0.01);而pcDNA3.1(+)/HPV6bL1(wt)组与载体和PBS组比较差异也有统计学意义(t=20.072、t=22.457,P值均＜0.01).在特异性CTL杀伤实验中,当E:T分别为5:1、10:1、20:1时,pcDNA3.1(+)/HPV6bL1(mod.)组的脾细胞特异性CTL杀伤率明显高于pcDNA3.1(+)/HPV6bLl(wt)组(t=11.892、t=15.329、t=2.911,P值均＜0.05)、载体组(t=12.936、t=16.613、t=13.90l,P值均＜0.01)和PBS对照组(t=14.768、t=18.935、t=10.925,P值均＜0.01).结论 密码子优化的HPV6b L1 DNA可诱导BALB/c小鼠产生增强的特异性体液和细胞免疫应答.     

条件性免疫反应治疗胶原性关节炎的免疫学机制探讨

目的探讨条件免疫反应治疗胶原性关节炎(CIA)免疫学机制。方法建立大鼠CIA模型。将模型大鼠分为5组:条件免疫反应(CIR)组:以樟脑气味为条件刺激;甲氨蝶呤(MTX)+泼尼松(Pred)为非条件刺激,两者结合7次(7d)后可建立条件免疫反应,然后每日再现条件刺激,每周条件刺激与非条件刺激结合1次,共4周。MTX+Pred组:MTX+Pred治疗4周。MTX+Pred减量组:MTX+Pred治疗,7d内每天1次,7d后每周1次,共4周。单纯闻樟脑气味组:单纯闻樟脑气味4周。空白对照组:安慰剂治疗4周。用流式细胞技术检测大鼠外周血CD4+T淋巴细胞活化(表达CD71)状态,免疫散射比浊法检测大鼠外周血IgG、IgA、IgM、C3、C4、C反应蛋白(CRP)水平,免疫组织化学检测CD68(巨噬细胞标志)及肿瘤坏死因子(TNF)-α在滑膜组织的表达。结果CIR组与MTX+Pred组大鼠治疗2周后外周血活化的CD4+T淋巴细胞明显低于其他各组(P<0.01);4周后血清IgG、CRP水平降低,滑膜组织中CD68及TNF-α表达均明显低于单纯闻樟脑气味组、MTX+Pred减量组及空白对照组(P均<0.01)。结论以樟脑气味为条件刺激,以泼尼松和甲氨蝶呤联合免疫抑制作用为非条件刺激,通过强化训练,建立的条件免疫抑制反应可以通过调节机体的细胞免疫与体液免疫功能,抑制免疫细胞异常活化,降低自身抗体及炎性细胞因子水平,     

Murine strain differences and the effects of zinc on cadmium concentrations in tissues after acute cadmium exposure

The role of strain differences in cadmium tissue distribution was studied using sensitive (129/J) and resistant (A/J) mice. These murine strains have previously been shown to differ in their susceptibility to cadmium-induced testicular toxicity. Cadmium concentration was measured in testis, epididymis, seminal vesicle, liver, and kidney at 24 h after cadmium chloride exposure (4, 10, and 20 μmol/kg CdCl₂). The 129/J mice exhibited a significant increase in cadmium concentration in testis, epididymis, and seminal vesicle at all cadmium doses used, compared to A/J mice. However, cadmium concentrations in liver and kidney were not different between the strains, at any dose, indicating that cadmium uptake is similar in these organs at 24 h. These murine strains demonstrate similar hepatic and renal cadmium uptake but significantly different cadmium accumulation in the reproductive organs at 24 h. The mechanism of the protective effect of zinc on cadmium toxicity was studied by assessing the impact of zinc acetate (ZnAc) treatment on cadmium concentrations in 129/J mice after 24 h. Zinc pretreatment (250 μmol/kg ZnAc), given 24 h prior to 20 μmol/kg CdCl₂ administration, significantly decreased the amount of cadmium in the testis, epididymis, and seminal vesicle of 129/J mice, and significantly increased the cadmium content of the liver after 24 h. Cadmium levels in the kidney were unaffected at this time. Zinc pretreatment also prevented the cadmium-induced decrease in testicular sperm concentration and epididymal sperm motility seen in 129/J mice. These findings suggest that the differences in the two murine strains may be attributed partly to the differential accumulation of cadmium in murine gonads. This may be caused by strain differences in the specificity of cadmium transport mechanisms. The protective role of zinc in cadmium-induced testicular toxicity in the sensitive strain may be due to an interference in the cadmium uptake by susceptible reproductive organs. Received: 8 June 1998 / Accepted: 18 August 1998     

Effect of maternal cadmium exposure on postnatal development and tissue cadmium,copper and zinc concentrations in rats

Administration of 60 ppm cadmium (Cd) in drinking water from the 1^st to the 20^th day of gestation to female rats did not affect the viability, body weight gain, food, and water consumption of offspring. The blood hemoglobin level was reduced in 2-week-old females and males but not in 16-week-old offspring. Hematocrit and serum glucose level were not affected at either age. Cadmium concentration in the intestinal wall was increased in both age groups, with marginal uptake in other organs. A decrease in copper (Cu) concentration was found in the brain of 2-week-old offspring of both sexes and of 16-week-old females. The brain zinc (Zn) concentration was decreased only in 16-week-old animals. The physical and neuromuscular development of offspring before weaning was not impaired by maternal Cd treatment. The alterations in Cu and Zn metabolism were associated with reduced locomotor activity and affected open-field behavior in adult offspring of either sex and with decreased avoidance acquisition in adult female offspring.The results obtained suggest a relationship between the reduced brain Cu and Zn levels and CNS dysfunction in adult offspring of female rats exposed to Cd during gestation.     

Effects of cadmium exposure on thyroid gland and endochondral ossification in Rana zhenhaiensis

Discovery of elevated concentrations of cadmium in the natural environment has increased awareness because of their potential threats. Amphibians are negatively affected due to their moderate sensitivity to cadmium. Here, we conduct acute and subchronic toxicity tests to examine whether, and to what extent, cadmium exposure disturbs metamorphosis, growth, and kinetic ability of Rana zhenhaiensis. We set different concentration treatment groups for the subchronic toxicity test (0, 10, 40, 160 μg Cd L⁻¹). Our findings demonstrate that cadmium exposure reduces growth parameters and the cumulative metamorphosis percent of R. zhenhaiensis. Decreases in follicular size and follicular epithelial cell thickness of thyroid gland are found in the treatment group. Further, subchronic exposure to cadmium decreases ossification ratio of hindlimbs in all treatment. Also, adverse effects of cadmium exposure on aquatic tadpoles can result in the reduced physical parameters and weak jumping ability in adult frogs. In this sense, our study suggests that cadmium adversely influences body condition and metamorphosis of R. zhenhaiensis, damages thyroid gland and impairs endochondral ossification. Meanwhile, we speculated that cadmium-damaged thyroid hormones inhibit skeletal development, resulting in the poor jumping ability, which probably leads to reduced survival of R. zhenhaiensis.     

Effects of cadmium exposure on zinc and copper distribution in neonatal rats

Tissue zinc and copper concentrations undergo marked changes in the neonatal rat during the first several weeks of life and it was of considerable interest to study the effect of cadmium exposure on these ontogenic changes. Long evans rats received either 2 or 10 mol cadmium chloride per kg SC at 9 days of age and were sacrificed at 20 or 36 days of age. Tissue copper and zinc concentrations in cadmium-treated rats were compared to those of age-matched controls for statistically significant changes. The tissue affected, the element altered and the direction of change in concentration, increased (+) or decreased (-), are summarized for the two dosing groups (age at dosing, age at sacrifice in days): 2 mol/kg (9, 20): kidney Zn (+), blood Zn (-), cerebral Cu (-), cerebellar Cu (+); 2 mol/kg (9, 36): blood Zn (-); 10 mol/kg (9, 20); liver, kidney, cerebral and cerebellar and blood Zn (-), cerebellar Cu (+); 10 mol/kg (9, 36]: liver and heart Zn (+), blood Zn (-); liver and heart Zn (+), blood Zn (-); kidney, cerebral, cerebellar and heart Cu (+). Changes in tissue zinc or copper concentrations produced by cadmium treatment could not be accounted for by the direct replacement of these elements by cadmium and may be due to alterations in transport of these elements. These results indicate that early life exposure to low levels of camium can have large and persistent effects on the distribution of the essential metals, copper and zinc.     

肾病综合征患儿细胞免疫功能的检测及其意义

目的 探讨肾病综合征(NS)患儿细胞免疫功能的变化及其意义.方法 应用流式细胞仪检测45例NS患儿及30例健康对照组外周血T淋巴细胞亚群及自然杀伤细胞(NK)的水平.结果 NS患儿活动期CD3+、CD4+、CD4+/CD8+比值、NK[CD(16+56)]+细胞均明显低于Ns缓解期和对照组(P<0.05),缓解期CD4+细胞明显低于对照组(P<0.05).结论 NS的发病与细胞免疫功能紊乱密切相关,这为调节患儿免疫功能的相关治疗提供一定依据.     

Microarray analysis of changes in bone cell gene expression early after cadmium gavage in mice

We developed an in vivo model for cadmium-induced bone loss in which mice excrete bone mineral in feces beginning 8 h after cadmium gavage. Female mice of three strains [CF1, MTN (metallothionein-wild-type), and MT1,2KO (MT1,2-deficient)] were placed on a low-calcium diet for 2 weeks. Each mouse was gavaged with 200 microg Cd or vehicle only. Fecal calcium was monitored daily for 9 days, beginning 4 days before cadmium gavage, to document the bone response. For CF1 mice, bones were taken from four groups: +/- Cd, 2 h after Cd and +/- Cd, 4 h after Cd. MTN and MT1,2KO strains had two groups each: +/-Cd, 4 h after Cd. PolyA+ RNA preparations from marrow-free shafts of femura and tibiae of each +/- Cd pair were submitted to Incyte Genomics for microarray analysis. Fecal Ca results showed that bone calcium excreted after cadmium differed for the three mouse strains: CF1, 0.24 +/- 0.08 mg; MTN, 0.92 +/- 0.22 mg; and MT1,2KO, 1.7 +/- 0.4 mg. Gene array results showed that nearly all arrayed genes were unaffected by cadmium. However, MT1 and MT2 had Cd+/Cd- expression ratios >1 in all four groups, while all ratios for MT3 were essentially 1, showing specificity. Both probes for MAPK 14 (p38 MAPK) had expression ratios >1, while no other MAPK responded to cadmium. Vacuolar proton pump ATPase and integrin alpha v (osteoclast genes), transferrin receptor, and src-like adaptor protein genes were stimulated by Cd; other src-related genes were unaffected. Genes for bone formation, stress response, growth factors, and signaling molecules showed little or no response to cadmium. Results support the hypothesis that Cd stimulates bone demineralization via a p38 MAPK pathway involving osteoclast activation.     

Effects of cadmium exposure during pregnancy on cadmium and zinc concentrations in neonatal liver and consequences for the offspring

The effects of cadmium exposure during pregnancy (by means of daily subcutaneous injections of 4.4 mol/kg to the mother) on the neonates were investigated. No effect was observed on fetal or neonatal body weights, nor on neonatal liver weights. These parameters were examined up to 5 weeks after birth. The weight of neonatal thymuses was decreased 7 and 14 days after birth due to cadmium exposure of the mothers as compared with controls. This may be caused by zinc deficiency, because zinc concentrations in fetal and neonatal livers after cadmium exposure were found to be very low 20 days after conception and 5 h after birth. Cadmium concentration in neonatal liver decreased; however, cadmium in malignant liver increased as age increased. In the mother, cadmium was transferred to the milk, as it was demonstrated in the stomach contents of the pups. Simultaneous administration of zinc in amounts equimolar to cadmium did not have any noticeable effect on the amount of cadmium transferred to the fetus or on cadmium concentrations in any of the organs investigated. It could not prevent zinc deficiency in fetal and neonatal liver. In addition, growth retardation of the thymus from exposed pups could not be prevented by zinc administration.     

Recovery of mouse embryos after short-term in vitro exposure to toxic nickel chloride

The development of preimplantation mouse embryos in vitro was adversely affected by the addition of nickel chloride (NiCl₂·6H₂O) to the culture medium. For day 3 (4–8 cell) embryos developmental cessation occurred after 48 h in culture, in NiCl₂·6H₂O-containing medium. However, transfer to NiCl₂·6H₂O-free medium after 5 min, 1 h, and 3 h exposure, resulted in regaining of the developmental capacity for a proportion of the exposed embryos.The in vivo development, in pseudopregnant recipients, of in vitro nickel-exposed embryos was not significantly different from that in control embryos.The results indicated that the effect of NiCl₂·6H₂O on the development of day 3 mouse embryos in vitro was reversible after a short exposure period.     

Pulmonary macrophage phagocytosis and enzyme production after in vivo exposure to silica dust

Guinea pigs were exposed to silica dust (SiO2) aerosols at 6, 13, 28, and 46 mg/m3 for 3 weeks and examined 4, 8, 16, and 23 weeks later. Twenty-four milligrams of TiO2 per cubic meter served as the control dust. Lung weights were increased at 8 weeks and later. Free lung cells showed a tendency to decrease in phagocytosis capacity at 8 weeks after cessation of exposure and later. Alveolar macrophage production of N-acetyl-beta-D-glucosaminidase, cathepsin D, and acid phosphatase was decreased at 8 weeks after exposure and later. The relationship between the depressant effect on macrophages and the absence of an exposure-related polymorphonuclear neutrophil response for the development of fibrosis could be part of the mechanism behind fibrosis.     

N-乙酰半胱氨酸和亚硒酸钠对镉亚慢性毒性的影响

目的探讨N-乙酰半胱氨酸(N-acetyl cysteine,NAC)和亚硒酸钠(Na2SeO3)对亚慢性染镉大鼠肝肾毒性的影响及其机制。方法32只Wistart大鼠随机分为4组,每组8只,第1组为对照组,第2组为单位染镉组,第3、4组为干预组。大鼠连续6周皮下注射7μmol/kg氯化镉,然后干预组分别腹腔注射1 mmol/kg NAC和10μmol/kg Na2SeO3,共2周;测定大鼠尿N-乙酰-β-苷酶(NAG)、碱性磷酸酶活力(ALP)和肝、肾皮质谷胱甘肽(GSH)、丙二醛(MDA)含量及谷胱甘肽过氧化物酶(GSH-Px)活力。结果亚慢性染镉使大鼠肝、肾皮质和尿镉含量显著升高,尿ALP、NAG和蛋白含量显著升高,肝、肾皮质GSH含量显著升高,GSH-Px活力显著降低。与单纯染镉组比较,NAC处理组尿镉、NAG和蛋白含量显著下降,肝、肾皮质GSH显著降低;Na2SeO3处理组尿镉、ALP及肝、肾皮质GSH含量显著下降,GSH-Px活力显著升高。结论NAC和Na2SeO3对镉致肾损伤的恢复具有促进作用,其机制可能与NAC或Na2SeO3改变体内GSH含量和GSH-Px活力有关。     

Effects of cadmium on cell death and cell proliferation in chick embryos

The aim of this study was to examine cell death and cell proliferation in chick embryos destined to have ventral body wall defects as a result of cadmium (Cd) treatment. Embryos in shell-less culture were treated with 50 μL Cd acetate (8.9 × 10⁻⁵ M Cd²⁺) at Hamilton–Hamburger (H.–H.) stage 16–17, or with equimolar sodium acetate. TdT-Mediated dUTP nick end labelling (TUNEL) showed the mode of cell death to be apoptosis commencing 4 h after treatment in somites and neural tube. Desquamation also occurred in the peridermal layer of the ectoderm. Cd caused no changes in the S-phase population of any tissue except ectoderm. The peridermal layer of the latter had a 40% reduction in labeling index (LI) 5.25 h after treatment but increased thereafter, being 30% greater than control values at 25.25 h. The occurrence of gross malformation was strongly correlated with the degree of apoptosis and in turn with the extent of peridermal desquamation. Pre-treatment with zinc acetate (10× the dose of Cd) prevented gross malformation, apoptosis and the effect of Cd on peridermal proliferation. We hypothesize that the ventral body wall defect resulting from Cd treatment in chick embryos is the result of changes in the somites perhaps following interruption of a signalling pathway originating in ectoderm.     

Activity of glutathione-S-transferase in rat liver and kidneys after administration of lead or cadmium

The influence of an acute dose of lead nitrate or cadmium chloride on the activity of glutathione-S-trans-ferase (GST) was investigated in rats. CdCl₂ (10 mol/kg) did not influence the enzyme activity in either organ. In contrast Pb(NO₃)₂ (100 mol/kg) caused a significant increase of GST activity in both organs. SDS polyacrylamide gel electrophoresis (SDS-PAGE) showed that in liver the activity enhancement is due mainly to the induction of the isoenzyme GST 7-7, while in the kidneys the activity of all the isoenzymes is increased.     

Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose

Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo.     

Maternal exposure to arsenic and cadmium and the risk of congenital heart defects in offspring

Hair arsenic and cadmium from 339 women with congenital heart defect (CHD)-affected pregnancies (case women) and 333 women with normal live births (control women) in China were estimated using inductively coupled plasma mass spectrometry. The median levels of hair arsenic and cadmium in the case women were 98.30 (74.30–136.30) ng/g and 14.60 (8.30–32.50) ng/g, respectively, which were significantly higher than the levels in the control group (P < 0.05). Arsenic concentrations ≥62.03 ng/g were associated with increased risk for almost every CHD subtype, with a dose-response relationship. However, only the group with the highest cadmium levels (≥25.85 ng/g) displayed an increased risk of CHDs (AOR 1.96; 95% CI 1.24–3.09), with a 2.81-fold increase found for the occurrence of conotruncal defects in their offspring. Furthermore, an interaction between arsenic and cadmium was observed. Our findings suggest that maternal exposure to arsenic and cadmium may be a significant risk factor for CHDs in offspring. Cadmium may have an enhancing effect on the association between arsenic and the risk of CHDs in offspring.     

Binding of cadmium and mercury by metallothionein in the kidneys and liver of rats following repeated administration

Rats were injected subcutaneously with either cadmium chloride (0.5 mg Cd/kg) or mercuric chloride (0.5 and 0.25 mg Hg/kg) every other day over a period of 6 to 7 weeks. Levels of metals and metallothionein were determined in liver and kidneys. Both cadmium and metallothionein accumulated in these organs during the period of exposure. Mercury and metallothionein accumulated only in the kidneys. Mercury did not accumulate in the liver, nor did it cause an increase of metallothionein in the liver. The molar ratio of 1.6 (metal to metallothionein) in liver was constant for cadmium. In the kidneys this ratio varied from 1 to 3 for both metals, depending on the level of the metal; This ratio was 0.1 for liver mercury.This work was partly supported by the Polish-American agreement No. 05-009-2 with National Institute for Occupational Safety and Health, PHS, USA.     

LYRM03联合多黏菌素B治疗小鼠肺炎克雷伯菌肺炎的药效学研究

目的 将新化合物LYRM03和多黏菌素B联合用于治疗小鼠肺炎,拟观察在达到同等治疗效果的情况下,联合LYRM03是否能够降低多黏菌素B的用量。方法 采用耳窥镜直视下气管插管法建立小鼠肺炎模型,采用多黏菌素B单药或联合LYRM03的不同给药方案进行治疗,观察期96h,从小鼠存活率、体重变化、活动度评分、肺组织细菌载量等指标比较两者的治疗效果。结果 高剂量多黏菌素B治疗组疗效明显优于低剂量组,但单药与联合用药间无明显差别。多黏菌素B按1和5mg/kg单药治疗组存活率分别为50%和100%,联合用药后存活率分别为50%和83.3%。单药与联合用药治疗后两组间其余各项观察指标也无显著性差异。结论 联合LYRM03与多黏菌素B单药治疗相比,疗效没有明显差异,提示联合LYRM03不能降低多黏菌素B的用量。     

Behavioral alterations in offspring of female rats repeatedly exposed to cadmium oxide by inhalation

Prolonged exposure of female rats to cadmium oxide aerosols (0.02 and 0.16 mg Cd/m3) in air had no effect on fertility. Viability and postnatal growth of the offspring of dams that were exposed to 0.16 mg Cd/m3 before and during gestation, however, were depressed. Forepaw muscular strength and endurance of pups in all groups were similar. Maternal Cd exposure resulted in reduction of exploratory motor activity in 3-month-old pups from the 0.16 mg Cd/m3 group and male offspring from the 0.02 mg Cd/m3 group. Dose-dependent decreases of avoidance acquisition were seen in female offspring but not in males. In the open-field test, the ambulation of 5-month-old males from the 0.16 mg Cd/m3 was lowered, whereas in females from the 0.02 mg Cd/m3 group it was enhanced. The results indicate central nervous system (CNS) dysfunction in offspring of female rats exposed to low concentrations of cadmium oxide by inhalation.