The assessment of genotoxic effects in lymphocyte cultures of infants treated with chloral hydrate

Chloral hydrate is a sedative commonly used in pediatric medicine. It was evaluated for genotoxicity in cultured peripheral blood lymphocytes of infants who were given chloral hydrate for sedation. Sister chromatid exchange and micronucleus frequencies were determined before and after chloral hydrate administration. After treatment, the frequencies of sister chromatid exchange and micronuclei were significantly increased, suggesting that chloral hydrate has moderate genotoxic potential in infants.     

Notes on Technic

Fixation in 20 to 40% pyridin or in 10% chloral hydrate followed by 10 to 40% pyridin gave the most consistent staining of pericellular structures in the spinal cord of cat. Chloral hydrate perfusion and soaking followed by ammoniated alcohol (Hoff's application of Cajal's method) was uniformly successful only when pyrogallol instead of hydroquinone was used as a reducing agent. Perfusion of the animal with chloral hydrate gave a rather questionable degree of improvement over fixation by simple soaking. The difficulty in selecting a routine procedure as the “best” became apparent when no single experimental variation was outstandingly superior in all animals.     

Block Staining of Nervous Tissue with Silver: III. Pericellular end-bulbs or boutons

Fixation in 20 to 40% pyridin or in 10% chloral hydrate followed by 10 to 40% pyridin gave the most consistent staining of pericellular structures in the spinal cord of cat. Chloral hydrate perfusion and soaking followed by ammoniated alcohol (Hoff's application of Cajal's method) was uniformly successful only when pyrogallol instead of hydroquinone was used as a reducing agent. Perfusion of the animal with chloral hydrate gave a rather questionable degree of improvement over fixation by simple soaking. The difficulty in selecting a routine procedure as the “best” became apparent when no single experimental variation was outstandingly superior in all animals.     

Immunofluorescent staining of kinetochores in micronuclei: a new assay for the detection of aneuploidy

The immunofluorescent staining of kinetochores in micronuclei with antikinetochore antibodies was used to develop an in vitro assay for aneuploidy-inducing agents. The results show that about 80% of micronuclei induced by either colchicine or chloral hydrate contained kinetochores; only 9% of X-ray-induced micronuclei reacted positively to the antibody. These findings indicate that the in vitro micronucleus assay coupled with immunofluorescent staining of kinetochores can be a useful method for assessing the ability of chemicals to induce aneuploidy and/or chromosome aberrations.     

Characterization of cytotoxic and genotoxic effects of different compounds in CHO K5 cells with the comet assay (single-cell gel electrophoresis assay)

Different variants of the comet assay were used to study the genotoxic and cytotoxic properties of the following eight compounds: chloral hydrate, colchicine, hydroquinone, DL-menthol, mitomycin C, sodium iodoacetate, thimerosal and valinomycin. Colchicine, mitomycin C, sodium iodoacetate and thimerosal induced genotoxic effects. The other compounds were found to be inactive. The compounds were tested in the standard comet assay as well as in the all cell comet assay (recovery of floating cells after treatment), designed in our laboratory for adherently-growing cells. This latter procedure proved to be more adequate for the assessment of the cytotoxicity for some of the compounds tested (hydroquinone, DL-menthol, thimerosal, valinomycin). Colchicine was positive in the standard comet assay (3h treatment) and in the all cell comet assay (24h treatment). Sodium iodoacetate and thimerosal were positive in the standard and/or the all cell comet assay. Chloral hydrate, hydroquinone, sodium iodoacetate, mitomycin C and thimerosal were also tested in the modified comet assay using lysed cells. Mitomycin C and thimerosal showed effects in this assay, whereas sodium iodoacetate was inactive. This indicates that it does not induce direct DNA damage. Compounds that are known or suspected to form DNA-DNA cross-links or DNA-protein cross-links (chloral hydrate, hydroquinone, mitomycin C and thimerosal) were checked for their ability to reduce ethyl methanesulfonate (EMS)-induced DNA damage. This mode of action could be demonstrated for mitomycin C only.     

Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.     

Mutagenicity of methyl 2-benzimidazolecarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies and micronuclei

Methyl 2-benzimidazolecarbamate (MBC), diethylstilbestrol (DES) and estradiol were tested with regard to their ability to induce C-mitoses, polyploidies, micronuclei, structural chromosomal aberrations and sister-chromatid exchanges (SCE) in human peripheral lymphocytes in vitro. The compounds did not induce structural chromosomal aberrations either in the presence or absence of metabolic activation. MBC and estradiol were negative in the SCE test. DES induced SCE rates which were not even twice the control level and which were independent of dose and of metabolic activation. All compounds induced C-mitoses, polyploidies and micronuclei. The micronuclei are interpreted as resulting from errors in the anaphase distribution of chromosomes by spindle disturbances rather than from structural chromosomal aberrations.     

Spontaneous and induced activation of rat oocytes

Ovulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase-like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase--the two processes that normally occur in succession as a response to oocyte activatin--can be experimentally separated.     

Differential effects of chloral hydrate- and ketamine/xylazine-induced anesthesia by the s.c. route

The proper use of anesthetics in animal experimentation has been intensively studied. In this study we compared the use of chloral hydrate (500 mg kg(-1)) and ketamine (167 mg kg(-1)) combined with xylazine (33 mg kg(-1)) by the s.c. route in male Wistar rats. Chloral hydrate and ketamine/xylazine produced a depth of anesthesia and analgesia sufficient for surgical procedures. The decrease of systolic and diastolic blood pressure was of a higher magnitude in rats anesthetized with chloral hydrate than with ketamine/xylazine. The initial microvascular diameter and blood flow velocity did not differ between both agents. On the other hand, ketamine/xylazine reduced the heart rate more intensively than chloral hydrate. Both anesthetics promoted an increase in arterial pCO(2) and a decrease in pH levels compared to unanesthetized animals. The blood glucose levels were of a higher magnitude in rats after ketamine/xylazine anesthesia than after chloral hydrate. In mesenteric arterioles studied in vivo, ketamine/xylazine anesthesia reduced the constrictive effect of noradrenaline and the dilator effect of bradykinin. However, both anesthetics did not modify the vasodilator effect promoted by acetylcholine. Based on our data, we concluded that both anesthetics alter metabolic and hemodynamic parameters, however the use of chloral hydrate in studies of microvascular reactivity in vivo is more appropriate since ketamine/xylazine reduces the responses to vasoactive agents and increases blood glucose levels.     

水合氯醛、乌拉坦及其1:1 混合液在SD 大鼠麻醉中的效果比较及应用

目的:比较水合氯醛、乌拉坦及其1:1混合液在SD大鼠麻醉中的效果并进一步在大鼠模型制备的麻醉中检验其效果。方法:分别采用不同剂量的水合氯醛和乌拉坦及其1:1混合液进行麻醉实验,比较其麻醉起效时间、维持时间和死亡率,并将相同剂量的1:1混合液应用于SD大鼠模型制作时的麻醉中,比较其与非模型组之间的差异。结果:水合氯醛和乌拉坦混合液麻醉大鼠的起效时间2.5±1.5分钟,与单用水合氯醛无差异(P>0.05),比单用乌拉坦起效时间短(P<0.05);维持时间107.4±4.1分钟,比单用水合氯醛、乌拉坦长(P<0.01);麻醉死亡率比单用水合氯醛低,总死亡率比单用水合氯醛、乌拉坦低。模型组大鼠的麻醉起效时间2.9±1.6分钟,维持时间108.9±4.4分钟,零麻醉死亡率,总死亡率为2.5%;与1:1混合液非模型组的麻醉效果没有明显差异。结论:水合氯醛+乌拉坦1:1混合液麻醉效果好、起效快、死亡率极低,适合用于2小时左右的SD大鼠手术或模型制作。     

Bioactivation of nitroglycerin by purified mitochondrial and cytosolic aldehyde dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC(50) values of 42 +/- 2.9 and 3.1 +/- 0.4 microm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.     

1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline-induced apoptosis in the human neuroblastoma cell line SK-N-SH

Trichloroethylene, a common industrial solvent and a metabolic precursor of chloral hydrate, occurs widely in the environment. Chloral hydrate, which is also used as a hypnotic, has been found to condense spontaneously with tryptamine, in vivo, to give rise to a highly unpolar 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) that has a structural analogy to the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Earlier studies have revealed the relative permeability of the molecule through the blood-brain barrier and its ability to induce Parkinson-like symptoms in rats. In this study, we report that TaClo induces an apoptotic pathway in the human neuroblastoma cell line, SK-N-SH, involving the translocation of mitochondrial cytochrome c to the cytosol and activation of caspase 3. TaClo-induced apoptosis shows considerable differences from that mediated by other Parkinson-inducing agents such as MPTP, rotenone and manganese. Although it is not clear if the clinically administered dosage of chloral hydrate or the relatively high environmental levels of trichloroethylene could lead to an onset of Parkinson's disease, the spontaneous in vivo formation of TaClo and its pro-apoptotic properties, as shown in this report, should be considered.     

水合氯醛、乌拉坦及其1：1混合液在SD大鼠麻醉中的效果比较及应用

目的：比较水合氯醛、乌拉坦及其1：1混合液在SD大鼠麻醉中的效果并进一步在大鼠模型制备的麻醉中检验其效果。方法：分别采用不同剂量的水合氯醛和乌拉坦及其1：1混合液进行麻醉实验,比较其麻醉起效时间、维持时间和死亡率,并将相同剂量的1：1混合液应用于SD大鼠模型制作时的麻醉中,比较其与非模型组之间的差异。结果：水合氯醛和乌拉坦混合液麻醉大鼠的起效时间2.5±1.5分钟,与单用水合氯醛无差异（P〉0.05）,比单用乌拉坦起效时间短（P〈0.05）;维持时间107.4±4.1分钟,比单用水合氯醛、乌拉坦长（P〈0.01）;麻醉死亡率比单用水合氯醛低,总死亡率比单用水合氯醛、乌拉坦低。模型组大鼠的麻醉起效时间2.9±1.6分钟,维持时间108.9±4.4分钟,零麻醉死亡率,总死亡率为2.5%;与1：1混合液非模型组的麻醉效果没有明显差异。结论：水合氯醛＋乌拉坦1：1混合液麻醉效果好、起效快、死亡率极低,适合用于2小时左右的SD大鼠手术或模型制作。     

Metabolic activation of hydroquinone by macrophage peroxidase

Lysates from macrophages, cells involved in hematopoiesis and immunological responses, catalyzed the metabolic activation of the benzene metabolite, hydroquinone, to protein-binding compounds and to free 1,4-benzoquinone. This reaction is mediated by a peroxidase since activation was dependent upon H2O2 and was prevented by the inhibitors aminotriazole and azide. Activation of hydroquinone was independent of HO. radicals since protein binding occurred in the presence of the HO. scavengers mannitol and dimethyl sulfoxide. In reactions with macrophage lysates, phenol, another hepatic metabolite of benzene, stimulated the production of 1,4-benzoquinone as well as the amount of hydroquinone equivalents bound to protein in a dose-dependent manner. Addition of cysteine to incubations with macrophage lysates resulted in a dose-dependent decrease in hydroquinone equivalents bound to protein. At 100 microM cysteine, protein binding was inhibited by 63% and this decrease was recovered as the monocysteine-hydroquinone conjugate. Macrophages catalyzed the arachidonic acid-mediated activation of hydroquinone to metabolites which bound to cellular macromolecules. This activation was inhibited by indomethacin indicating the action of prostaglandin synthase in hydroquinone metabolism by macrophages. The results of these experiments demonstrate that macrophage peroxidase catalyzes the metabolic oxidation of hydroquinone to 1,4-benzoquinone and that 1,4-benzoquinone and/or its semiquinone intermediate are binding to protein and cysteine. Hydroquinone activation by macrophages and subsequent macromolecular binding may be associated with the immunologic and hematopoietic toxicity of benzene.     

The Effects of Apomorphine upon Local Cerebral Glucose Utilization in Conscious Rats and in Rats Anesthetized with Chloral Hydrate

The effects of the dopaminergic agonist apomorphine (1 mg . kg-1 i.v.) upon local cerebral glucose utilization in 43 anatomically discrete regions of the CNS were examined in conscious, lightly restrained rats and in rats anesthetized with chloral hydrate by means of the quantitative autoradiographic [14C]2-deoxyglucose technique. In animals anesthetized with chloral hydrate, glucose utilization was reduced throughout all regions of the CNS from the levels observed in conscious animals, although the magnitude of the reductions in glucose use displayed considerable regional heterogeneity. With chloral hydrate anesthesia, the proportionately most marked reductions in glucose use (by 40-60% from conscious levels) were noted in primary auditory nuclei, thalmaic relay nuclei, and neocortex, and the least pronounced reductions in glucose use (by 15-25% from conscious levels) were observed in limbic areas, some motor relay nuclei, and white matter. In conscious, lightly restrained rats, the administration of apomorphine (1 mg . kg-1) effected significant increased in glucose utilization in 15 regions of the CNS (e.g., subthalamic nucleus, ventral thalamic nucleus, rostral neocortex, substantia nigra, pars reticulata), and significant reductions in glucose utilization in two regions of the CNS (lateral habenular nucleus and anterior cingulate cortex). In rats anesthetized with chloral hydrate, the effects of apomorphine upon local glucose utilization were less widespread and less marked than in conscious animals. In only two of the regions (the globus pallidus and septal nucleus), which displayed increased glucose use following apomorphine in conscious rats, were significant increases in local glucose utilization observed with this agent in chloral hydrate-anesthetized rats. In the pars compacta of the substantia nigra, in which apomorphine increased glucose utilization in conscious animals, significant reductions in glucose utilization were observed following apomorphine in rats anesthetized with chloral hydrate. The profound effects of chloral hydrate anesthesia upon local cerebral glucose use, and the modification by this anesthetic regime of the local metabolic responses to apomorphine, emphasize the difficulties which exists in the extrapolation of data from anesthetized animals to the conditions which prevail in the conscious animal.     

Effect of Chloral Hydrate on Methane and Propionic Acid in the Rumen

Methane production from pyruvate by mixed rumen bacteria in vitro was nearly totally inhibited by chloral hydrate (0.1 mumole/ml of incubation fluid). This effect was accompanied by an accumulation of gaseous hydrogen and an increase in propionic acid production. Infusion of chloral hydrate (4 g/day) into the rumen of a sheep produced the same effects. Evidence is presented for a direct toxic effect of chloral hydrate upon methane bacteria. Results are discussed in terms of fermentation balances.     

Fate of 2,2,2-trichloroacetaldehyde (chloral hydrate) produced during trichloroethylene oxidation by methanotrophs.

Four different methanotrophs expressing soluble methane monooxygenase produced 2,2,2-trichloroacetaldehyde, or chloral hydrate, a controlled substance, during the oxidation of trichloroethylene. Chloral hydrate concentrations decreased in these cultures between 1 h and 24 h of incubation. Chloral hydrate was shown to be biologically transformed to trichloroethanol and trichloroacetic acid by Methylosinus trichosporium OB3b. At elevated pH and temperature, chloral hydrate readily decomposed and chloroform and formic acid were detected as products.     

Fate of 2,2,2-trichloroacetaldehyde (chloral hydrate) produced during trichloroethylene oxidation by methanotrophs.

Four different methanotrophs expressing soluble methane monooxygenase produced 2,2,2-trichloroacetaldehyde, or chloral hydrate, a controlled substance, during the oxidation of trichloroethylene. Chloral hydrate concentrations decreased in these cultures between 1 h and 24 h of incubation. Chloral hydrate was shown to be biologically transformed to trichloroethanol and trichloroacetic acid by Methylosinus trichosporium OB3b. At elevated pH and temperature, chloral hydrate readily decomposed and chloroform and formic acid were detected as products.     

Effect of anesthetics on efficiency of remote ischemic preconditioning

Remote ischemic preconditioning of hind limbs (RIPC) is an effective method for preventing brain injury resulting from ischemia. However, in numerous studies RIPC has been used on the background of administered anesthetics, which also could exhibit neuroprotective properties. Therefore, investigation of the signaling pathways triggered by RIPC and the effect of anesthetics is important. In this study, we explored the effect of anesthetics (chloral hydrate and Zoletil) on the ability of RIPC to protect the brain from injury caused by ischemia and reperfusion. We found that RIPC without anesthesia resulted in statistically significant decrease in neurological deficit 24 h after ischemia, but did not affect the volume of brain injury. Administration of chloral hydrate or Zoletil one day prior to brain ischemia produced a preconditioning effect by their own, decreasing the degree of neurological deficit and lowering the volume of infarct with the use of Zoletil. The protective effects observed after RIPC with chloral hydrate or Zoletil were similar to those observed when only the respective anesthetic was used. RIPC was accompanied by significant increase in the level of brain proteins associated with the induction of ischemic tolerance such as pGSK-3β, BDNF, and HSP70. However, Zoletil did not affect the level of these proteins 24 h after injection, and chloral hydrate caused increase of only pGSK-3β. We conclude that RIPC, chloral hydrate, and Zoletil produce a significant neuroprotective effect, but the simultaneous use of anesthetics with RIPC does not enhance the degree of neuroprotection.     

On the contribution of the acrylate pathway to the formation of propionate from lactate in the rumen of cattle

The effect of chloral hydrate, an inhibitor of methanogenesis, on the participation of the acrylate pathway in the formation of propionate from lactate in rumen contents of cattle was studied in vitro. Addition of chloral hydrate resulted in only a small stimulation of the acrylate pathway, much lower than the stimulation of propionate production by chloral hydrate. This means that the flux of carbon through both the acrylate and the dicarboxylic acid pathway is increased during chloral hydrate feeding.The influence of time of sampling after feeding on the contribution of the acrylate pathway was studied in a separate experiment. A marked drop in the participation of the acrylate pathway in propionate formation from lactate during at least 2 h after feeding was observed, whereafter a rapid rise to prefeeding levels occurred.