Coronary artery hypoxic vasorelaxation is augmented by perivascular adipose tissue through a mechanism involving hydrogen sulphide and cystathionine-ß-synthase.

AIM: Hypoxia causes vasodilatation of coronary arteries which protects the heart from ischaemic damage through mechanisms including the generation of hydrogen sulphide (H2 S), but the influence of the perivascular adipose tissue (PVAT) and myocardium is incompletely understood. This study aimed to determine whether PVAT and the myocardium modulate the coronary artery hypoxic response and whether this involves hydrogen sulphide. METHODS: Porcine left circumflex coronary arteries were prepared as cleaned segments and with PVAT intact, myocardium intact or both PVAT and myocardium intact, and contractility investigated using isometric tension recording. Immunoblotting was used to measure levels of H2 S-synthesizing enzymes: cystathionine-ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (MPST). RESULTS: All three H2 S-synthesizing enzymes were detected in the artery and myocardium, but only CBS and MPST were detected in PVAT. Hypoxia elicited a biphasic response in cleaned artery segments consisting of transient contraction followed by prolonged relaxation. In arteries with PVAT intact, hypoxic contraction was attenuated and relaxation augmented. In arteries with myocardium intact, hypoxic contraction was attenuated, but relaxation was unaffected. In replacement experiments, replacement of dissected PVAT and myocardium attenuated artery contraction and augmented relaxation to hypoxia, mimicking the effect of in situ PVAT and indicating involvement of a diffusible factor(s). In arteries with intact PVAT, augmentation of hypoxic relaxation was reversed by amino-oxyacetate (CBS inhibitor), but not DL-propargylglycine (CSE inhibitor) or aspartate (inhibits MPST pathway). CONCLUSION: PVAT augments hypoxic relaxation of coronary arteries through a mechanism involving H2 S and CBS, pointing to an important role in regulation of coronary blood flow during hypoxia.

A critical role for cystathionine-ß-synthase in hydrogen sulfide-mediated hypoxic relaxation of the coronary artery.

Hypoxia-induced coronary artery vasodilatation protects the heart by increasing blood flow under ischemic conditions, however its mechanism is not fully elucidated. Hydrogen sulfide (H2S) is reported to be an oxygen sensor/transducer in the vasculature. The present study aimed to identify and characterise the role of H2S in the hypoxic response of the coronary artery, and to define the H2S synthetic enzymes involved. Immunoblotting and immunohistochemistry showed expression of all three H2S-producing enzymes, cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST), in porcine coronary artery. Artery segments were mounted for isometric tension recording; hypoxia caused a transient endothelium-dependent contraction followed by prolonged endothelium-independent relaxation. The CBS inhibitor amino-oxyacetate (AOAA) reduced both phases of the hypoxic response. The CSE inhibitor dl-propargylglycine (PPG) and aspartate (limits MPST) had no effect alone, but when applied together with AOAA the hypoxic relaxation response was further reduced. Exogenous H2S (Na2S and NaHS) produced concentration-dependent contraction followed by prolonged relaxation. Responses to both hypoxia and exogenous H2S were dependent on the endothelium, NO, cGMP, K+ channels and Cl-/HCO3- exchange. H2S production in coronary arteries was blocked by CBS inhibition (AOAA), but not by CSE inhibition (PPG). These data show that H2S is an endogenous mediator of the hypoxic response in coronary arteries. Of the three H2S-producing enzymes, CBS, expressed in the vascular smooth muscle, appears to be the most important for H2S generated during hypoxic relaxation of the coronary artery. A contribution from other H2S-producing enzymes only becomes apparent when CBS activity is inhibited.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria.

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques. Simvastatin, but not pravastatin, produced a slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10µM) and the complex III inhibitor myxothiazol (10µM), or a combination of the two. The complex III inhibitor antimycin A (10µM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10µM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone. In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel.

Carbamazepine toxic effects in chick cardiomyocyte micromass culture and embryonic stem cell derived cardiomyocyte systems--possible protective role of antioxidants.

The use of carbamazepine (CBZ) during pregnancy increases cardiovascular anomalies. In this study CBZ developmental cardiotoxic effects were evaluated using chick cardiomyocyte micromass (MM) culture and mouse embryonic stem cells derived cardiomyocyte (ESDC) systems. In MM culture, CBZ only inhibited the cardiomyocyte contractile activity, while in ESDC it completely ceased the contractile activity at 200 µM with decreased cell viability and protein content. The antioxidant superoxide dismutase (SOD) supplement in MM and ascorbic acid (AA) in ESDC showed protective effects on CBZ toxicity, but elevated levels of reactive oxygen species (ROS) production were recorded with CBZ treatment only in ESDC. CBZ has also affected cardiac connexin 43 expression in both in vitro systems. Our results indicated CBZ induced ROS stress as mechanism of developmental cardiotoxicity at early stage of cardiogenesis in ESDC system compared to MM system's differentiated cells. These toxic effects can be negated by using antioxidant agent.

A role for the sodium pump in H2O2-induced vasorelaxation in porcine isolated coronary arteries.

Hydrogen peroxide (H2O2) has been proposed to act as a factor for endothelium-derived hyperpolarization (EDH) and EDH may act as a 'back up' system to compensate the loss of the NO pathway. Here, the mechanism of action of H2O2 in porcine isolated coronary arteries (PCAs) was investigated. Distal PCAs were mounted in a wire myograph and pre-contracted with U46619 (1nM-50µM), a thromboxane A2-mimetic or KCl (60mM). Concentration-response curves to H2O2(1µM-1mM), bradykinin (0.01nM-1µM), sodium nitroprusside (SNP) (10nM-10µM), verapamil (1nM-10µM), KCl (0-20mM) or Ca(2+)-reintroduction (1µM-10mM) were constructed in the presence of various inhibitors. Activity of the Na(+)/K(+)-pump was measured through rubidium-uptake using atomic absorption spectrophotometry. H2O2 caused concentration-dependent vasorelaxations with a maximum relaxation (Rmax) of 100±16% (mean±SEM), pEC50=4.18±0.20 (n=4) which were significantly inhibited by PEG-catalase at 0.1-1.0mM H2O2 (P<0.05). 10mM TEA significantly inhibited the relaxation up to 100µM H2O2 (P<0.05). 60mM K(+) and 500nM ouabain significantly inhibited H2O2-induced vasorelaxation producing a relaxation of 40.8±8.5% (n=5) and 47.5±8.6% (n=6) respectively at 1mM H2O2 (P<0.0001). H2O2-induced vasorelaxation was unaffected by the removal of endothelium, inhibition of NO, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa Kir, KV, KATP or cGMP. 100µM H2O2 had no effects on the KCl-induced vasorelaxation or Ca(2+)-reintroduction contraction. 1mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na(+)/K(+)-pump activity. We have shown that the vascular actions of H2O2 are sensitive to ouabain and high concentrations of H2O2 are able to modulate the Na(+)/K(+)-pump. This may contribute towards its vascular actions.

Hydrogen sulphide-induced relaxation of porcine peripheral bronchioles.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H2S) is an endogenous gasotransmitter. Although it has been shown to elicit responses in vascular and other smooth muscle preparations, a role for endogenously produced H2S in mediating airway tone has yet to be demonstrated. Therefore, the aim of this study was to determine whether H2S is produced within the airways and to determine the functional effect on airway tone. EXPERIMENTAL APPROACH: Small peripheral airways (<5 mm in diameter) from porcine lungs were set up in isolated tissue baths, pre-contracted with the muscarinic agonist carbachol, and then exposed to either the H2S donor sodium hydrosulphide (NaHS), or the precursor L-cysteine. H2S production from L-cysteine or 3-mercaptopyruvate in tissue homogenates was measured by the methylene blue assay. Expression of the H2S-synthesizing enzymes cystathionine ß-synthase (CBS), cystathionine γ lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (3-MST) were measured by Western blotting. KEY RESULTS: NaHS caused a large relaxation of the airways, which was inhibited partially by pre-contraction with KCl or exposure to tetraethylammonium, but not glibenclamide, paxilline or 4-aminopyridine. L-cysteine also caused a relaxation of the airways which was inhibited by the CBS inhibitor aminooxyacetic acid. Tissue homogenates from airways exposed to L-cysteine or 3-mercaptopyruvate in vitro showed a significant production of H2S. Western blotting demonstrated immunoreactivity to CBS, CSE and 3-MST enzymes in the airways. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that H2S can be produced endogenously within porcine airways causing relaxation. The mechanism of relaxation depends, in part, on K(+) channel activity.

Examination of the effect of procalcitonin on human leucocytes and the porcine isolated coronary artery.

BACKGROUND: The aim of this study was to investigate the effects of procalcitonin on the lipopolysaccharide (LPS)-induced changes in human leucocytes and porcine isolated coronary artery. METHODS: Using flow cytometry, changes in forward scatter and intracellular calcium in human neutrophils and monocytes were determined after exposure to procalcitonin, calcitonin gene-related peptide (CGRP), LPS, and the known chemoattractants formylated methionine-leucine-phenylalanine (fMLP) and interleukin-8 (IL-8). In porcine isolated coronary artery, the effects of procalcitonin were evaluated using the contractile function change and the release of TNFalpha. RESULTS: In human neutrophils and monocytes, procalcitonin (100 nM), but not CGRP, increased forward scatter and the expression of surface markers (CD16 and CD14, respectively) in a similar manner to 10 microg ml(-1) LPS. Procalcitonin, but not CGRP, also increased the proportion of cells exhibiting an increase in intracellular calcium ions similar to that produced by fMLP and IL-8. Acute exposure of the coronary artery to procalcitonin produced a small, endothelium-independent relaxation (approximately 15% of constrictor tone), but failed to modify subsequent relaxations to CGRP. After 16 h exposure, procalcitonin (100 nM) increased TNFalpha release from the coronary artery equivalent to 70% of that produced by LPS, but did not modify the inhibitory effect of LPS (100 microg ml(-1)) on contractile responses. CONCLUSIONS: Procalcitonin has a proinflammatory effect on human leucocytes and porcine coronary artery, but it is not capable of modulating LPS-induced changes in vascular responsiveness in vitro.

Cannabinoid activation of PPAR alpha; a novel neuroprotective mechanism.

BACKGROUND AND PURPOSE: Although CB(1) receptor activation evokes neuroprotection in response to cannabinoids, some cannabinoids have been reported to be peroxisome proliferator activated receptor (PPAR) ligands, offering an alternative protective mechanism. We have, therefore, investigated the ability of a range of cannabinoids to activate PPAR alpha and for N-oleoylethanolamine (OEA), an endogenous cannabinoid-like compound (ECL), to evoke neuroprotection. EXPERIMENTAL APPROACH: Assays of PPAR alpha occupancy and gene transactivation potential were conducted in cell-free and transfected HeLa cell preparations, respectively. In vivo estimates of PPAR alpha activation through fat mobilization and gene transcription were conducted in mice. Neuroprotection in vivo was investigated in wild-type and PPAR alpha gene-disrupted mice. KEY RESULTS: The ECLs OEA, anandamide, noladin ether and virodhamine were found to bind to the purified PPAR alpha ligand binding domain and to increase PPAR alpha-driven transcriptional activity. The high affinity synthetic CB(1/2) cannabinoid agonist WIN 55212-2 bound to PPAR alpha equipotently with the PPARalpha agonist fenofibrate, and stimulated PPARalpha-mediated gene transcription. The phytocannabinoid delta 9 tetrahydrocannabinol was without effect. OEA and WIN 55212-2 induced lipolysis in vivo, while OEA pre-treatment reduced infarct volume from middle cerebral artery occlusion in wild-type, but not in PPAR alpha-null mice. OEA treatment also led to increased expression of the NFkappa B-inhibitory protein, Ikappa B, in mouse cerebral cortex, while expression of the NFkappa B-regulated protein COX-2 was inhibited. CONCLUSIONS AND IMPLICATIONS: These data demonstrate the potential for a range of cannabinoid compounds, of diverse structures, to activate PPAR alpha and suggest that at least some of the neuroprotective properties of these agents could be mediated by nuclear receptor activation.

Modulation of hepatocyte thiol content by medium composition: implications for toxicity studies.

Toxicity of compounds requiring glutathione for detoxification, thiol content and synthesis were determined in 24-h rat hepatocytes cultured in medium containing different concentrations of the sulphur amino acids. Glutathione synthesis was determined following prior depletion of glutathione with diethylmaleate. L-15 medium, which has high levels of cysteine and methionine (1 mM of each), provided some protection against dichloroacetone, dibromopropanol and dichloropropanol toxicity, and had a small effect on increasing glutathione content and synthesis, relative to Williams' medium E (WE) which has low levels (less than 0.5 mM) of both amino acids. However, WE containing N-acetylcysteine (NAC) (1 mM final cysteine concentration), with or without methionine (final concentration 1 mM), was a better cytoprotectant medium than L-15, markedly reducing toxicity of all three compounds, and rapidly (within 1.5 h) increasing cellular glutathione content. WE supplemented with methionine alone stimulated glutathione synthesis after an initial lag phase, and protected cultures against dichloropropanol, but not dibromopropanol or dichloroacetone, both of which are highly reactive in these cultures. There was a clear association between glutathione content at early time points in culture and toxicity observed at later time points, and overall these results indicate that differences in culture medium composition can alter intracellular glutathione content and xenobiotic toxicity.

Workplace drug testing (WDT) likely to increase in Europe. Report from the First European Symposium on WDT including selected abstracts.

Will it take a series of drug-related accidents that have already occurred in the USA before workplace drug testing (WDT) becomes accepted in Europe as a preventive measure? Currently, the development of WDT in most European countries lags some 10-15 years behind that in the USA. Labour authorities in Europe now ought to take initiatives to demand a mandatory programme for accrediting drug analytical laboratories for WDT. Companies should realise that illicit drug use is no longer only a problem at street corners, and that having a testing system in place is important, not just for public health, but also for their reputations as responsible societal actors. Improved networking among police and regulatory authorities is required to keep pace with the rapid appearance and dissemination of new substances of abuse. European research collaboration, including the newly formed European Workplace Drug Testing Group, is needed to assess the impact of drug-testing policies on accidents and other outcome variables, and thereby to convince the general public and politicians that drug testing is beneficial and necessary. A 1993-1994 survey of quality analysis in some 200 European laboratories reported from Institut Municipal d'Investigació Medica (IMIM), Spain, showed good agreement between nominal and found concentrations but that only 10% of the laboratories could both screen, identify and quantify samples. Experiences from Italy show that proficiency testing schemes lead to improved accuracy of results. These were some major conclusions of the First European Symposium on Drug Testing held at Huddinge University Hospital in Stockholm, Sweden, 30 March to 1 April 1998, organised by Karolinska Institute, with participants from 22 countries.

Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: lack of involvement of the vanilloid receptor.

In studies into the oxidative burst in RAW 264 monocyte/macrophages, it was observed that capsaicin, a vanilloid receptor agonist, stimulated dichlorofluorescin (DCFH) oxidation in a concentration-dependent manner, which could be blocked by capsazepine, a vanilloid receptor antagonist. However, by use of a number of vanilloid agonists (including N-octyl-3-chloro-4-hydroxyphenylacetamide, 4m), we demonstrated that there was no relationship between vanilloid agonist potency and the capacity to stimulate DCFH oxidation. The oxidative burst stimulators Tween 20 and phorbol myristyl acetate (PMA) also stimulated reactive oxygen species generation, which again was inhibited by capsazepine. Use of the selective inhibitor diphenyliodonium iodide ruled out a role for plasma membrane NAD(P)H oxidase as the site of capsaicin- and 4m-stimulated DCFH oxidation. However, this DCFH oxidation was modulated by a number of inhibitors of mitochondrial respiration. Rotenone enhanced DCFH oxidation induced by capsaicin and 4m, whilst malonic acid and potassium cyanide inhibited this response. 2,4-Dinitrophenol, an inhibitor of oxidative phosphorylation, was without effect. The antioxidant trolox c inhibited DCFH oxidation stimulated by capsaicin, 4m, and PMA, whereas N-acetylcysteine, a precursor of glutathione, was without effect. Capsazepine inhibited DCFH oxidation in unstimulated cells and in cells treated with menadione, a redox-cycling quinone. Capsazepine was also a potent antioxidant when measured in a Fe3+ reduction assay. We concluded that DCFH oxidation stimulated by vanilloid analogues was not mediated via a vanilloid receptor, but rather by impairment of mitochondrial electron transport.

Haloalcohols deplete glutathione when incubated with fortified liver fractions.

1. This study has examined the ability of dichloropropanols, haloalcohols and their putative metabolites to deplete glutathione when incubated with liver fractions obtained from untreated and differentially induced rats. 2. 1,3-Dichloropropan-2-ol and 2,3-dichloropropan-1-ol (0-1000 microM) both depleted glutathione in a dose-dependent manner when incubated with cofactors (NADPH generating system) and liver microsomes from the untreated rat. 3. The extent of GSH depletion was significantly enhanced when liver microsomes from the isoniazid- or isosafrole-treated rat were used. 4. Epichlorohydrin produced a moderate, dose-dependent depletion of GSH. By contrast, 1,3-dichloroacetone (identified by TLC as a metabolite of 1,3-dichloropropanol) was a potent depletor of glutathione. 5. N-acetylcysteine was less efficient than glutathione as a nucleophile trap for epichlorohydrin, 1,3-dichloroacetone or reactive metabolites derived from 1,3-dichloropropan-2-ol. 6. 1,3-Dibromopropan-2-ol and 1,4-dibromobutan-2-ol were potent depletors of GSH but 1-bromopropan-2-ol produced less GSH depletion. Both dibromoalcohols depleted GSH when incubated with dialysed cytosol derived from the livers of untreated rats. 7. The GSH depletion mediated by 1,3-dichloropropan-2-ol, 1,3-dibromopropan-2-ol, 1,4-dibromobutan-2-ol and 1-bromopropan-2-ol was inhibited by inclusion of pyridine (1 mM) or cofactor omission. 1,3-Difluoropropanol did not deplete GSH under any of the conditions examined.

The nature of halogen substitution determines the mode of cytotoxicity of halopropanols.

The cytochrome P450-dependent generation of reactive metabolites from 1,3-dichloropropanol and 1,3-dibromopropanol was assessed in a microsomal thiol depletion assay, while the toxicity of these compounds was assessed in rat hepatocyte cultures and in the 3T3 cell line. Thiol-depleting metabolites of both compounds were generated in the microsomal assay; however, only dibromopropanol extensively depleted glutathione when glutathione S-transferase was used as the enzyme source. The cytotoxicity of dichloropropanol was both cytochrome P450- and glutathione-dependent, whereas that of dibromopropanol was glutathione-dependent but largely independent of cytochrome P450. These results indicate that the mechanisms underlying the cytotoxicity of halopropanols are dependent on the nature of the halogen substitution and that microsomal and cellular assays for reactive metabolite generation may yield conflicting results.

A Preliminary Comparison of LiverBeads™ with a Conventional Rat Hepatocyte Culture Preparation: Some Aspects of Xenobiotic Metabolism and Related Toxicity.

LiverBeads™ are made of hepatocytes that are immobilised and cryopreserved in alginate gel. They have great potential as an easily transportable and easily handled source of hepatocytes for use in in vitro pharmacotoxicology. In this study, we compared the drug metabolising capacity of LiverBeads in culture with that of conventional cultures and of cultures derived from cryopreserved cells. Trypan blue exclusion, lactate dehydrogenase and DNA content were measured in LiverBead cultures. The levels were all similar to those of the conventional cultures, as were the toxicities of precocene II and allyl alcohol, although more variability was seen in the LiverBeads than in the conventional cultures. The cytochrome P450-dependent activity 3,4-dimethyl-7-ethoxycoumarin-O-dealkylase, was reduced in the LiverBeads when compared to the conventional cultures, although the pattern of conjugation was very similar. In addition, the inducibility of cytochrome P4504A was demonstrated in LiverBeads. Cultures from cryopreserved cells were more susceptible to the toxicants tested, and contained less lactate dehydrogenase and DNA than the conventional cultures. Overall, in terms of the aspects of drug metabolism measured, the cultures from LiverBeads appeared to be equivalent to conventional cultures, and superior to cultures from cryopreserved cells.

The Use of Human Keratinocytes in the EU/COLIPA International In Vitro Phototoxicity Test Validation Study and the ECVAM/COLIPA Study on UV Filter Chemicals.

The EU/COLIPA in vitro phototoxicity study involved the testing of 30 chemicals in Phase II, and the ECVAM/COLIPA study on UV filter chemicals involved the testing of 20 chemicals, for which in vivo human and/or animal data were available. Primary human keratinocytes, from four separate male donors, were not found to be sensitive to the 5J/cm2 UVA produced by the SOL500 lamp when assayed by using the neutral red uptake endpoint, as employed with the 3T3 cells used in these international interlaboratory validation studies. The primary human keratinocytes tested in one laboratory alongside the 3T3 fibroblasts gave consistent indications of phototoxicity with all the phototoxicants tested in the Phase II and UV filter studies. The one exception was bithionol, which was predicted to be non-phototoxic in both studies. None of the non-phototoxic chemicals resulted in a positive reaction with the Photoirritation Factor (PIF) version of the prediction model. However, when the Mean Photo Effect (MPE) prediction model version was applied (with a cut-off point of 0.1), one sunscreen agent, octyl salicylate, was deemed to have phototoxic potential. The entire set of negative rated chemicals included in Phase II and in the UV filter study were also rated as non-phototoxic by the MPE prediction model.

Imipramine for Cytochrome P450 Activity Determination: a Multiple-species Metabolic Probe.

Imipramine is metabolized in vitro by rat, mouse or human microsomes to several metabolites including 2-hydroxyimipramine, 2-hydroxydesipramine, desipramine, didesmethylimipramine and imipramine-N-oxide. Heat treatment (which inhibits FMO), verified the role of this group of enzymes in the oxidation of imipramine to imipramine N-oxide. These studies demonstrate that while rats and humans showed similar metabolic profiles, producing several minor metabolites in addition to the 2-hydroxyimipramne and desipramine, differences were seen in the mouse microsomes. In these samples, few minor metabolites were produced. In addition, imipramine N-oxide was the major metabolite in mouse, whereas none was detected in human samples. To conclude, the metabolic profile of imipramine can be used to reveal a number of cytochrome P450 enzymes active in microsomal fractions.

Fluorescein cadaverine incorporation as a novel technique for the characterization of terminal differentiation in keratinocytes.

A novel technique for detecting transglutaminase activity and the production of cornified envelopes in keratinocytes has been devised. This was based on the enzymatic incorporation of fluorescein-labelled cadaverine (FC) into cornified envelopes. The addition of FC (20mum) to the incubation medium served as an amine donor for transglutaminase reactions in place of protein lysine residues. Cells incorporating the label became visible with fluorescence microscopy and were quantified by fluorimetry. There was a significant difference in the level of FC incorporation into cornified envelopes under the various media conditions and time points employed. The greatest incorporation was observed by keratinocytes cultured in Green's medium and fluorescent intensity decreased in the order: Green's> KGM with calcium>KGM. Confocal imaging of keratinocytes dual stained with FC and propidium iodide revealed the presence of distinct layers and demonstrated how FC was incorporated into differentiating cells and not the basal layer. FC incorporation has the potential to serve as a rapid assessment of terminal differentiation in keratinocytes. It is simple and less time-consuming than currently available alternative techniques. This approach also has the advantage of combining microscopic and quantitative data.

Anabolic steroids and violent crime--an epidemiological study at a jail in Stockholm, Sweden.

Violent crime has been associated with the abuse of anabolic-androgenic steroids (AAS) in several reports. Speculations concerning such associations have been raised with regard to several recent crimes committed in Sweden. To test this hypothetical relationship, individuals in a Stockholm jail who had been arrested for violent crimes were screened for AAS in the urine. No AAS were detected in the urine samples of 50 prisoners who had volunteered for the study. However, 16 prisoners refused to participate. AAS abuse was admitted by two of the participating subjects. Although there is a great need for epidemiological studies to objectively confirm the association of AAS abuse and violence, it seems that such studies will be impossible to conduct as long as they, for legal reasons, depend on voluntary participation.

Human keratinocyte cultures in an in vitro approach for the assessment of surfactant-induced irritation.

A specific, mechanistic, in vitro approach for the assessment of human skin irritation potential is outlined for the evaluation of surfactants and the results compared with in vivo human patch test data. The level of free available surfactant monomer and the solubilization of the corn protein zein in vitro were confirmed to be related to surfactant in vivo human skin irritation potential. In vitro cytotoxicity to monolayer keratinocyte cultures could not discriminate between the moderate human skin irritant sodium dodecyl sulfate (SDS) and the mild irritants cocamidopropylbetaine (CA) and Polysorbate 20 (P20). An in vitro stratified differentiated human epidermal equivalent (HEE) exhibited reduced cytotoxicity to the test chemicals, compared with monolayer culture responses, and was able to discriminate between the toxic potential of SDS and CA. Stimulation of interleukin-1alpha release from the A431 human keratinocyte cell line reflected in vivo erythema scores more closely than cytotoxic potential, and coincided with nitric oxide production by macrophages upon exposure to A431-conditioned medium. Combination of these mechanistic assays has allowed a profile of likely in vivo human responses to be approximated. Additional knowledge of skin penetrability and rate of recovery from toxic damage would affirm these predictions.

Increased urinary testosterone/epitestosterone ratios found in Swedish athletes in connection with a national control program. Evaluation of 28 cases.

In connection with a national anti-doping control program, including analysis of 8946 urine samples, 28 athletes were found to have delivered samples free from xenobiotic anabolic steroids but with an increased testosterone/epitestosterone (T/E) ratio (> 6). Unannounced testing of the above athletes produced 2-4 additional urine samples during the next 2-3 months. A low degree of variation of the T/E ratio, with a C.V. below 30% was found in 17 of the subjects whereas 10 had a C.V. varying from 31% to 43%. One subject with a high urinary T/E ratio (10.5) had a C.V. of this ratio of 126% and also an extremely high ratio between testosterone and LH in urine. It has been reported that non-users of testosterone have T/E ratios fluctuating around a mean with a C.V. that will not exceed 30%. We found that administration of testosterone to seven healthy volunteers resulted in urinary T/E ratios that varied with a C.V. ranging from 67% to 130% during the following 4 weeks. It is concluded that among the above 28 cases, only one can be regarded as a clear case of testosterone doping. Although the vast majority of Swedish athletes have urinary T/E ratios below six, there is a subfraction with a constant higher ratio, possibly due to genetic factors.