The influence of gender and experience on intubation ability and technique: a manikin study.

Female anaesthetists in early training commonly question whether their strength is adequate for tracheal intubation. This study investigated the influence of gender and experience on intubation ability and laryngoscopic technique. A manikin model and purpose-designed force-transducing laryngoscope was used to test three cohorts at different levels of experience (novice, intermediate and experienced males and females, n = 65) for the axial force and torque exerted, best laryngoscopic view obtained, success with and time for intubation and laryngoscopic technique. There were no significant differences between novice or experienced female and male intubators in markers of their ability to intubate or in the forces generated. For novice females compared with novice males, mean success rate was 90% (80.2 to 99.9) versus 97% (91.1 to 100, P = 0.29); and mean time to intubate 24 seconds (19 to 29 seconds) versus 18 seconds (14 to 21 seconds, P = 0.057). With experience, the forces generated during intubation reduced and ability improved. Proximal laryngoscope grips (close to the blade) generated lower forces than distal grips. Female and male intubators did not differ in ability to intubate or in the forces they exerted during direct laryngoscopy.

Cytotoxicity evaluation of anionic nanoliposomes and nanolipoplexes prepared by the heating method without employing volatile solvents and detergents.

Submicron lipid vesicles (nanoliposomes) are being used as carriers of bioactive compounds. In addition, complexes of nanoliposomes and nucleic acids (nanolipoplexes) are promising tools for the treatment of cancer, and viral and genetic disorders. Toxicity of some of these formulations, however, still remains a concern in their clinical utilisation. To address this problem, anionic liposomes were prepared by two different techniques, the conventional thin-film method, and the heating method (HM), in which no volatile organic solvent or detergent is used. An anionic nanolipoplex was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) into the HM-nanoliposomes by the mediation of calcium. The toxicity of the nanoliposomes, with and without plasmid and Ca2+, was assessed using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Cytotoxicity evaluations performed by two different assays (i.e. NRU and MTT) indicated that HM-nanoliposomes were completely non-toxic in the cell-line tested, whereas conventional liposomes revealed significant levels of toxicity. This may be due to the presence of trace amounts of chloroform and/or methanol applied during their preparation. Similar results were obtained for different sizes of lipid vesicles (prepared by 100 nm and 400 nm pore-size filters). In addition, it was observed that incorporation of DNA (15 microg/ 285 microg lipid) and Ca2+ (50 mM) to the nanoliposomes did not have any effect on their cytotoxicities. These findings indicate that the HM-liposomes have great potential as non-toxic delivery vehicles in human gene therapy and drug delivery applications while liposomes made using organic solvents should be used with caution.

A review of scanning probe microscopy investigations of liposome-DNA complexes.

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.

Induction of heat shock protein 70 in rat olfactory epithelium by toxic chemicals: in vitro and in vivo studies.

We have previously developed a rat nasal explant system for investigating upper respiratory tract toxicity, and the aims of this study were to determine whether heat shock protein (HSP) 70 is induced in this model following exposure to carbon tetrachloride (CCl4), dimethyl adipate (DMA), methyl iodide (CH3I) or paracetamol, and whether HSP70 can also be induced in the nasal cavity in vivo. Intracellular ATP was significantly depleted in ethmoturbinates incubated for 4 h with the toxins (0-100 mM; EC50 concentrations: CCl4 32 mM, DMA 3 mM, CH3I 1.5 mM, paracetamol 70 mM), but there was little induction of HSP70. Turbinates were then incubated for 1 h with CCl4 (5 mM), DMA (1.5 mM), CH3I (0.57 mM) or paracetamol (30 mM) and allowed to recover for up to 24 h. Treatment with CCl4, DMA or paracetamol resulted in 250-300% induction of HSP70. Male rats were administered a single oral dose of CCl4 (1600 mg/kg) and killed 16 h later. Degenerative lesions (epithelial undulation and hydropic vacuolation) were evident in the olfactory epithelium, and immunohistochemical analysis of HSP70 revealed increased staining in, or proximate to, areas of damage. Thus, HSP70 can be induced in the olfactory epithelium both in vitro and in vivo.

Foetal rat lung epithelial (FRLE) cells: alterations in cellular homeostasis and gene expression in response to etoposide, hydrogen peroxide and sodium butyrate.

Genomics technology offers a way of detecting the effects of a toxin on the expression of many genes in a single experiment. We have previously partially characterised a foetal rat lung epithelial (FRLE) cell line and shown that it is suitable for use in a pneumocytotoxicity screen. In this study, we wanted to ascertain whether we could use alterations in FRLE cell gene expression as a sensitive marker of cell stress. Sodium butyrate and etoposide were shown to arrest FRLE cell cycle at G0/G1 and G2/M phase of the cell cycle, respectively and this was associated with a decrease in the number of cells in culture. Following 24 h of culture both compounds caused a statistically significant increase in the mRNA levels of the cell cycle inhibitory protein, gadd153, whereas p21 was statistically altered by etoposide only. Hydrogen peroxide induced growth arrest at low concentrations (< or =250 microM) following 24 h of culture. We could not detect an increase in apoptosis or in the mRNA levels of the pro-apoptotic protein bax in FRLE cells following culture with hydrogen peroxide or etoposide. Thus, it was possible to correlate cellular perturbations in FRLE cells with alterations in gene expression, demonstrating that these cells are suitable for use in a toxicity screen.

Foetal rat lung epithelial (FRLE) cells: partial characterisation and response to pneumotoxins.

Cultured cell lines are routinely used for in vitro toxicity screens, reducing the requirement for animal studies during the development of new pharmaceutical, agrochemical and cosmetic products. The foetal rat lung epithelial (FRLE) cell line was originally derived from alveolar type II cells (ATII) of the lung. The aims of this study were to further characterise FRLE cells and investigate their potential for screening for pneumotoxins. The cells were found to have retained some of the features of their progenitor cells, namely the expression of cytokeratin proteins, specifically cytokeratin 18, and the ability to actively accumulate the non-selective contact herbicide paraquat. However, the cells have lost the ability to synthesise surfactant protein mRNA and no longer contain multiple lamellar bodies. Toxins that damage ATII cells in vivo (cadmium chloride, cobalt chloride and paraquat) were found to induce cytotoxicity in FRLE cells, as did the non-specific pneumotoxin nitrofurantoin, and hydrogen peroxide. However, the cells were less sensitive to the effects of compounds that require metabolic activation (1-nitronaphthalene, coumarin and butylated hydroxytoluene) and the hepatotoxin bromobenzene. Thus, FRLE cells appear to be a good in vitro model for monitoring the potential toxicity to ATII cells and could be used as an initial screen for pneumotoxicity.

A rat nasal epithelial model for predicting upper respiratory tract toxicity: in vivo-in vitro correlations.

An in vitro model of the rat nasal cavity has been used to compare the responses of nasal tissues in vitro, using loss of intracellular ATP and potassium as indices of toxicity, with the pathological changes occurring following in vivo exposure to four test compounds. Turbinates were incubated in vitro with the test compounds for 4 h, for 24 h or for 4 h followed by 20 h in fresh medium. Titanium dioxide caused little or no loss of ATP in either olfactory epithelium (OE) or respiratory epithelium (RE). Sodium carbonate decreased olfactory, but not respiratory ATP, while acetic acid and 3-methylindole markedly decreased ATP in both tissues. Intracellular potassium concentrations were generally affected to a lesser degree. In vivo, no morphological changes were observed in the nasal cavity following inhalation exposure to either titanium dioxide or sodium carbonate. Inhalation of acetic acid resulted in a very focal lesion in the RE of the dorsal meatus of level 1, while administration of 3-methylindole by intraperitoneal injection caused severe degeneration of OE. In further experiments olfactory turbinates were exposed to a range of concentrations (0-100 mM) of sodium carbonate, acetic acid and 3-methylindole for 4 h and ATP concentrations determined. Concentration-dependent decreases in ATP were observed for sodium carbonate and 3-methylindole, with EC(50) values estimated as 2.57 and 0.91 mM, respectively. Acetic acid only decreased ATP significantly at the 100-mM concentration. In summary, this in vitro model has predicted the nasal toxicity of several compounds, including both direct-acting agents (sodium carbonate, acetic acid) and one requiring metabolic activation (3-methylindole). However, the lack of airflow-dependent dosimetry, results in some lack of discrimination between the different regions of the nasal cavity and may make this model overly sensitive.

Apoptosis and cancer: strategies for integrating programmed cell death.

Virtually all human cells are endowed with the capacity to commit suicide using an evolutionarily conserved mechanism that involves activation of caspase-family cell death proteases. Caspase activation culminates in a cell death process known as "apoptosis." The activation of these intracellular proteases is carefully controlled through a delicate balance of anti- and pro-death proteins, serving to precisely regulate cell life span. Defects in the natural death pathway promote tumorigenesis by prolonging cell life span and hence cell accumulation. Low-grade B-cell malignancies, particularly follicular lymphoma and chronic lymphocytic leukemia (CLL) represent quintessential examples of human neoplasms characterized primarily by a problem with cell death rather than cell cycle. Because the cell suicide pathway is also required for tumor eradication by the immune system, anticancer drugs, and irradiation, cancer-associated defects in the cellular apoptosis machinery also play an important role in treatment failures. Monoclonal antibody-based therapies may provide opportunities to either bypass defects in apoptosis pathways or to activate latent apoptotic programs in cancer cells, particularly in lymphoid malignancies where tissue-specific antigens can be exploited for cell-selective activation of apoptosis. Recent knowledge about apoptosis pathways is reviewed, and some examples of opportunities for therapeutic intervention are discussed.

Methyl iodide toxicity in rat cerebellar granule cells in vitro: the role of glutathione.

The monohalomethane methyl iodide (MeI) is toxic to a number of organ systems including the central nervous system. Clinical symptoms of neurotoxicity suggest that the cerebellum is the target within the brain, and we have now modelled the toxicity of MeI in cultured rat cerebellar granule cells. Cytotoxicity is maximal 24 h after a 5 min exposure to MeI, and the EC50 for MeI under these conditions was calculated to be 1.6 mM. The glutathione S-transferase (GST) dependent metabolism of MeI was investigated in these cultures. There was a marked decrease in intracellular glutathione (GSH) 15 min after exposure to MeI, and GSH concentrations then increased, reaching 130% of control levels 7 h after exposure. To investigate the role of conjugation with GSH in the toxicity of MeI, GSH levels were modulated prior to exposure. Depletion of GSH exacerbated the cytotoxicity of MeI while provision of a bioavailable source of GSH was protective. Inclusion of antioxidants [vitamin E, butylated hydroxytoluene (BHT) or desferrioxamine mesylate (DF)] also protected against the cytotoxicity of MeI. Our in vitro data suggest that MeI is conjugated with GSH in the cerebellum, and the resulting extensive depletion of GSH may be the first step en route to toxicity, rendering the tissue susceptible to methylation and/or oxidative stress.

Amplification of a pseudogene cassette underlies euchromatic variation of 16p at the cytogenetic level.

Euchromatic imbalances at the cytogenetic level are usually associated with phenotypic consequences. Among the exceptions are euchromatic variants of chromosomes 8, 9, 15 and 16, which have each been reported in multiple unrelated families. In this paper, we present a new family and an unrelated individual who have euchromatic variants of 16p. Enhanced hybridisation to the extra material was found by using fluorescence in situ hybridisation with cosmids for both the 16p11.2-specific non-functional immunoglobin heavy chain segments and the pseudogenetic 16p11.2 creatine transporter region. Computerised measurement of the fluorescent signals was consistent with amplification of a pseudogene cassette comprising both these paralogous domains, which were originally transposed from 14q32.3 and Xq28, respectively. Amplification of pseudogenetic sequences is consistent with the normal phenotype in 36/46 carriers from the 18 families reported to date. Inconsistent phenotypic anomalies in the remaining 10 carriers probably reflect bias of ascertainment. These results are analogous to the amplification of the 15q11.2-specific pseudogene cassette in euchromatic variants of chromosome 15. They also suggest that the majority of established euchromatic variants are associated with variation in the copy number of sequences that have been dispersed between pericentromeric and telomeric loci over recent evolutionary time. We propose that constitutional cytogenetic amplification of this kind is part of a more widespread continuum of genomic flux affecting regions in which heterochromatin and euchromatin interpose. Euchromatic sequences that vary in a heterochromatic manner might usefully be termed "hemichromatic".

Investigations of the pathways of toxicity of methyl iodide in the rat nasal cavity.

The monohalomethane methyl iodide (MeI) is a site specific toxin within the nasal cavity of the rat, selectively damaging the olfactory epithelium (OE) whilst respiratory epithelium (RE) is spared. The aim of this study was to investigate the rates and routes of metabolism of MeI within the nasal cavity, in order to understand the reasons for the observed site-selectivity. Cytosolic glutathione S-transferases (GSTs) of both OE and RE catalysed the conjugation of MeI with glutathione (GSH), but rates were 4-fold higher in OE than RE. The product of this reaction was confirmed as S-methyl GSH. In both OE and liver the GST catalysing the conjugation of MeI was shown to belong to the theta class. No cytochrome P450-dependent oxidation of MeI to formaldehyde could be detected in incubations containing hepatic or olfactory microsomes. Intact nasal turbinates were incubated with [14C]-MeI, and a dose- and time-dependent covalent binding of MeI to olfactory protein was demonstrated. The rates of protein methylation were found to be similar in OE and RE. Thus the only parameter that correlates with the site-selectivity of the observed lesion is the rate of conjugation of MeI with GSH. Whether toxicity is due to production of a reactive metabolite or GSH depletion per se, remains to be elucidated.

The role of glutathione S-transferase- and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat.

The aim of this study was to investigate the role of metabolic activation in the olfactory toxicity of methyl iodide (MeI). Adult male rats were exposed via nose-only inhalation to 100 ppm MeI for 0-6 h, and non-protein sulphydryl (NP-SH) concentrations determined in selected tissues. Depletion of NP-SH occurred in all tissues, but was most marked and rapid in the respiratory epithelium of the nasal cavity and the kidney. Olfactory, lung and liver NP-SH levels were affected to a lesser extent, and those of the brain declined by only 20-30% over the whole time course. In order to modulate glutathione (GSH) status, animals were pre-treated with (1) phorone plus L-buthionine sulphoximine (BSO), which depleted NP-SH levels in all the tissues examined, or (2) the isopropyl ester of GSH (IP-GSH), which was shown to replenish NP-SH concentrations in all tissues except the liver of animals previously administered phorone. When animals were pre-treated with phorone plus BSO and then exposed to 100 ppm MeI for 2 h, there was a potentiation of the toxicity of MeI as judged by the clinical observations on the animals. In contrast, treatment with IP-GSH prior to and during exposure to MeI for 4 h afforded a marked protection to the olfactory epithelium. In order to inhibit cytochromes P450, animals were pre-treated with cobalt protoporphyrin IX. This decreased hepatic cytochrome P450 concentrations by > 90%, but when animals were then exposed to 100 ppm MeI for 4 h there was no effect on the severity of the olfactory lesion. These results indicate that conjugation of MeI with GSH is a detoxification rather than an activation pathway. Also, there is no major role for cytochrome P450-dependent oxidation in the development of the olfactory lesion.

Xenobiotic metabolizing enzymes of the kidney.

The kidney possesses most of the common xenobiotic metabolizing enzymes, and is thus able to make an important contribution to the body's metabolism of drugs and foreign compounds. An overview of the renal localization, catalytic activity, developmental regulation, induction, and sex and species differences for the key enzymes involved in phase I and phase II of xenobiotic metabolism is presented. In general, the catalytic activities of the various renal enzymes are lower than those of the liver, although there are exceptions, such as the enzymes involved in the processing of glutathione conjugates to their mercapturic acids. Xenobiotic metabolizing enzymes are not evenly distributed along the nephron; cytochromes P-450 and those enzymes involved in the conjugation of glutathione, glucuronic acid, or sulfate are primarily localized in the proximal tubules. However, some isozymes of cytochrome(s) P-450 and glutathione S-transferases are selectively localized in cells of the thick ascending limb and distal tubules, whereas prostaglandin H synthase is concentrated in the collecting ducts in the medulla. Thus, the proximal tubule, the principal site of xenobiotic biotransformation, is particularly susceptible to chemical insult, and the localization of prostaglandin synthase in the inner medulla and papilla may be a contributary factor to the toxicity produced by chemicals in this part of the nephron. Many of the enzymes discussed, in addition to metabolizing foreign compounds, have important endogenous functions in the kidney, such as the regulation of salt and water balance and the synthesis of vitamin D.

Regulation of rat olfactory glutathione S-transferase expression. Investigation of sex differences, induction, and ontogenesis.

The glutathione S-transferases (GSTs) of rat olfactory epithelium have been characterised with regard to sex differences, induction, and developmental regulation, and compared to those of the liver. Olfactory cytosolic GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was similar in both male and female animals, and there were no differences in subunit profile. Administration of trans-stilbene oxide and beta-naphthoflavone had no effect on olfactory GST activity with CDNB, although phenobarbitone treatment resulted in a small, but significant, increase in activity (130% compared to controls). HPLC analysis of subunit profiles indicated that all three agents induced olfactory subunit 1b and decreased subunit 6. The effect of age (3 to 84 days) on both cytosolic and microsomal CDNB activity was examined. In the liver, cytosolic activity was low at 3 days and climbed steadily to reach maximal levels around 28 days, but microsomal activity was relatively constant at all ages. Olfactory cytosolic activity was similar at all ages; microsomal activity was low until 21 days and then increased to reach a maximum at 56 days. Changes in individual cytosolic subunits were assessed by SDS-PAGE followed by immunoblotting. The significance of these results with regard to putative physiological roles for olfactory GSTs is discussed.

Olfactory toxicity of methyl iodide in the rat.

The monohalomethanes (methyl iodide, methyl bromide and methyl chloride) are widely used industrial methylating agents with pronounced acute and chronic toxicity in both experimental animals and man. Recently inhalation exposure of rats to methyl bromide has been shown to result in severe olfactory toxicity. This study examined the effects on the rat nasal cavity of inhalation of methyl iodide (100 ppm for 0.5-6 h), and demonstrated that methyl iodide is a more potent olfactory toxin than methyl bromide. Within the nasal cavity the olfactory epithelium was the principle target tissue, and it was only at high doses (600 ppm.h) that limited damage to transitional epithelium occurred. The squamous and respiratory epithelia were consistently unaffected. Within olfactory epithelium the sustentacular cells were the primary cellular target and damage to sensory cells appeared to be a secondary event. Methyl iodide induced olfactory damage was reversible, and 2 weeks after exposure almost complete repair had taken place.

Immunohistochemical localisation of six glutathione S-transferases within the nasal cavity of the rat.

Many xenobiotics induce lesions within the nasal cavity of experimental animals which are site specific. This site selectivity may be due to regional deposition within the nasal cavity and/or the localisation of biotransformation enzymes. We have developed methodology which allows immunohistochemical localisation of xenobiotic biotransformation enzymes in transverse sections of the rat nasal cavity identical to those normally taken for pathological examination. We report the application of this methodology to six isoenzymes of the glutathione S-transferases (GSTs). All six isoenzymes were predominantly located within olfactory epithelium covering the ethmoturbinates (levels III and IV) and extending forwards into the dorsal meatus (level II). Squamous and transitional epithelia showed little or no staining while respiratory epithelium was weakly stained. Within the respiratory epithelium only the ciliated columnar cells and, to a lesser extent, some of the seromucous glands contained GSTs. Within olfactory epithelium the sustentacular cells, basal cells and subepithelial glands all stained positive for GSTs. The different cell types of olfactory epithelium preferentially express different GST isoenzymes: 1-1 and 2-2 were predominantly located in the subepithelial glands; 3-3, 4-4 and 8-8 in sustentacular and basal cells; 7-7 in basal cells.

The characterization of glutathione S-transferases from rat olfactory epithelium.

The glutathione S-transferases (GSTs) of rat olfactory epithelium have been characterized with regard to substrate specificity and subunit composition and compared to those of the liver. The presence of cytosolic GST activity in rat olfactory epithelium was confirmed and, using 1-chloro-2,4-dinitrobenzene as substrate, was found to be approximately one-third that of the liver. Olfactory microsomal GST activity was greater than that of liver microsomes and could be activated by treatment with the sulphydryl agent N-ethylmaleimide. The subunit and isoenzyme profile of GSTs in the olfactory epithelium was investigated using a number of techniques. (1) Olfactory GSTs were characterized using a range of relatively subunit-specific substrates. Activities ranged from 40-90% of those found in liver. Most noticeable was the extremely low olfactory activity with the substrate specific for subunit 1. (2) Immunoblotting with antibodies against specific rat hepatic GSTs confirmed the presence of a number of subunits and the absence of subunit 1. (3) F.p.l.c. chromatofocusing and reverse-phase h.p.l.c. indicated that the cytosolic GST profile of olfactory epithelium is unique and is made up of subunits 2, 3, 4, 7, 8 and 11 with subunits 3 and 4 predominating. There is an absence of isoenzymes containing subunit 1.

Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission.

In the presence of Cu(II) ions, supercoiled DNA is cleaved in neutral solution by low concentrations of thiols. Supercoiled plasmid DNA is cleaved first to open circular DNA, which in turn produces linear DNA and eventually fragments. Cleavage is strongly temperature-dependent and is maximal at 0.10-0.25 M-NaCl concentration. In the presence of excess of either component of the Cu(II)-thiol pair, the extent of cleavage depended on the concentration of the limiting partner, and was easily detectable down to micromolar concentrations of limiting GSH. Scavengers of oxygen-derived species (such as hydrogen peroxide, superoxide radical ion and hydroxyl radical) indicated that the hydroxyl radical may be involved in the cleavage mechanism. DNA cleavage leads to some production of 2-thiobarbituric acid-reactive species and some of the cleavage sites, at least, had 5'-hydroxy and/or 3'-hydroxy groups. There was extensive base damage before cleavage. Studies with S1 nuclease indicated no gross sequence preference for Cu(II)-GSH cleavage of pSP64 plasmid DNA. The Cu(II)-thiol system did not appear to target special structural features in the DNA such as Z-DNA inserts, cruciform structures or left-handed (but non-Z) DNA. Cleavage might arise from a reagent generated either by the Cu(II)-thiol combination in free solution or by attack involving Cu(II) ions pre-bound to DNA. The attack of GSH plus Cu(II) ions on DNA may be a potential toxic lesion under physiological conditions unless special protective measures operate efficiently in the cell.