beta-Adrenoceptor blocking drugs and isoprenaline: central effects on cardiovascular parameters.

1. The central hypotensive activity of (+)- and (-)-propranolol (100 microgram), pindolol (100 microgram) and isoprenaline (1 and 4 microgram) injected intracerebroventricularly (i.c.v.) was studied in rats anaesthetized with urethane and chloralose. Blood pressure, cardiac output and heart rate were measured; systolic stroke volume and peripheral vascular resistance were calculated. 2. (+)- and (-)-Propranolol and pindolol induced a fall of blood pressure but (+)-propranolol was less active. The heart rate was reduced more by (-)-propranolol than by (+)-propranolol or (-)-pindolol. The decrease of systolic stroke volume was greater for (-)-propranolol and pindolol than for (+)-propranolol. Peripheral vascular resistance was reduced to the same level but with different time courses, (-)-propranolol having a longer effect than (+)-propranolol and pindolol. 3. Isoprenaline induced a hypotensive effect, while cardiac output and heart rate increased; the systolic stroke volume remained stable but peripheral vascular resistance was significantly decreased. 4. These results suggest that different central regulatory centres are involved in the control of cardiac function and peripheral vascular tone.

Effects of chronic beta-blocker treatment on catecholamine levels in spontaneously hypertensive rats.

In the present work the effects of a 55-day oral treatment with two beta-blocking agents (propranolol 40 mg/kg per day and S 2395 20 mg/kg per day) on the catecholamine (CA) content of central and peripheral structures were studied in spontaneously hypertensive rats (SHR). The concentrations of dopamine (DA), noradrenaline (NA) and adrenaline (A) in different structures dissected out from treated and control SHR were measured by a radioenzymatic method. At the peripheral level, no change in the concentration of NA (in the heart) or A (in the adrenal medulla) was observed. Propranolol increased the DA concentration in the C1 and C2 regions of the medulla oblongata and S 2395 increased the DA concentration only in the C2 region. In these two areas, the NA and A levels were unchanged. Both propranolol and S 2395 increased the DA, NA and A content in the locus coeruleus and in the anterior hypothalamus. On the contrary, there was no modification in the posterior hypothalamus. The anatomical specificity of these alterations of the CA levels suggests that they could be related to a specific action of beta-blockers on central catecholaminergic structures in SHR which might be linked to the antihypertensive effects of these drugs.

Protection by 4-amino-hex-5-enoic acid (gamma-vinyl GABA) and hypobaric hypoxia against kainic acid neurotoxicity.

The behavioral effects following IP injection of kainic acid in rats could be cancelled with hypobaric hypoxia (8% O2 corresponding to 7000 m altitude) or with IP injections of 7-amino-hex-5-enoic acid at a dose of 0.9 g/kg-1. This protection may result from increased accumulations of GABA in brain tissues.

Effect of tabernanthine on the turnover time of brain catecholamines in normal and hypobaric hypoxic rats.

The central effects of tabernanthine on noradrenaline and dopamine turnover times were studied in the hypothalamus, the striatum and the remainder of the brain of normal and hypobaric hypoxic rats, the latter state corresponding to 5,200m (410mm Hg) and 7,000m (320mm Hg). Catecholamine cerebral level were not modified by the drug in either instance. At normal atmospheric pressure the catecholamine turnover times were slightly decreased by tabernanthine. Hypobaric hypoxia alone increased noradrenaline and dopamine turnover times by inhibiting oxygen-dependent enzymes. Tabernanthine antagonized the effect of hypobaric hypoxia especially in dopaminergic areas. Antagonism was complete at 5,200m but only partial at 7,000m. This phenomenon may be related to the stimulatory properties of the drug.

Follicles play an important role in percutaneous absorption.

The relative importance of stratum corneum and follicles in percutaneous absorption is not fully understood. In order to quantitatively investigate the importance of the transappendageal route, we have previously developed a model of skin without follicles regrown dorsally on the hairless rat. Percutaneous absorption was compared, using a diffusion cell, in appendage-free skin relative to normal skin, and a predominant role of follicles for in vitro diffusion of [3H]hydrocortisone was noted. Results presented here of in vitro diffusion of tritiated hydrocortisone, niflumic acid, caffeine, and p-aminobenzoic acid, applied in acetone, confirm that appendageal diffusion is the major pathway in hairless rat skin. In the absence of follicles, the steady-state flux and the amounts diffusing in 24 or 48 h are 2-4 times lower than in normal skin. These results were confirmed in a second model in which diffusion of [3H]hydrocortisone was studied on skin samples taken one day after birth, at which time rat skin is still devoid of follicles, relative to five-day postnatal skin samples, in which follicles are fully developed. The steady-state flux and the total diffusion in 24 h were fivefold lower in follicle-free skin. These results support the view that follicles may have a far greater importance in percutaneous absorption than is generally assumed.

Cutaneous bioavailability in hairless rats of tretinoin in liposomes or gel.

Topical bioavailability of drugs incorporated in liposomes is not well known. We compared the skin penetration of tretinoin in liposomes and in a classical alcoholic gel. [3H]Phosphatidylcholine dipalmitoyl (DPPC) and [14C]tretinoin (0.14%) were incorporated in the phospholipidic phase of the liposomes, and [14C]tretinoin was incorporated in a gel for comparison. Skin absorption was studied in vitro with Franz cells. In vivo distribution in cutaneous structures was studied according to Schaefer's method. Liposomes impregnated the stratum corneum, with a partial dissociation between tretinoin and phosphatidyl-choline dipalmitoyl. In dermis, tretinoin diffused alone. Tretinoin release seemed to be controlled, and steady state was reached later with liposomes than with gel. This phenomenon was linked with a significantly reduced absorption (1.60% for liposomes versus 3.09% for the gel) and higher retention in epidermis (mainly stratum corneum) and dermis (41 and 13%, respectively, with liposomal form versus 18 and 8%, respectively, with gel form). This study clearly shows that, compared with the gel, the liposome formulation tends to improve the local effect of tretinoin in the skin and decrease the systemic absorption.

Importance of sebaceous glands in cutaneous penetration of an antiandrogen: target effect of liposomes.

The significance of the sebaceous gland pathway in the cutaneous permeation of an antiandrogen, 4-[3-(4-hydroxybutyl)-4,4-dimethyl -2,5-dioxo-1-imidazolidinyl]-2-(trifluoromethyl)benzonitrile (RU 58841), was studied with normal hairless rat skin and an induced scar hairless rat skin without sebaceous glands. RU 58841 was dissolved in an alcoholic solution and encapsulated in liposomes for comparison. After 24 h, the cumulative percentage of RU 58841 absorbed in vitro was 3-4-fold higher in the normal skin than in the scar skin; in the case of liposomes, the accumulation of the drug in the normal dermis was significantly higher than in the scar one. In the in vivo cutaneous distribution, the epidermis and dermis of the normal skin contained higher amounts of RU 58841 than the scar skin (ninefold with the solution and 16-fold with liposomes). An autoradiography study showed that with the solution, the drug was mainly localized in the stratum corneum/epidermis, and with the liposomes, the drug was mainly localized in the sebaceous glands. We concluded that the sebaceous glands constituted the main pathway for RU 58841. The alcoholic solution encouraged the localization of the drug into the stratum corneum, whereas liposomes targeted the sebaceous glands.

Effect of proteins on the assessment of surfactant cytotoxicity by an in vitro test: Possible correlations with in vivo data.

Four surfactants were studied for their cytotoxic effect on human fibroblasts in culture. The surfactants were sodium dodecyl sulphate (SDS; anionic), cetyltrimethylammonium bromide (CTAB; cationic) and two nonionic compounds, octylphenoxy polyethoxyethanol (Triton X100) and polyoxyethylene (20) sorbitan monooleate (Tween 80). For each surfactant, LD(100) values were determined with varying amounts of protein-foetal calf serum (FCS) or bovine serum albumin (BSA)-in the culture medium, to evaluate the influence of protein concentration on surfactant cytotoxicity. FCS, like BSA, acted as a detoxifying agent, increasing the LD(100) values especially for SDS and Tween 80. Dialysis experiments enabled this general decrease in cytotoxicity to be correlated with a binding process. The binding of surfactants to the proteins of the culture medium could also be estimated by calculation, using the LD(100) values, and the values obtained were found to be similar to those determined by the dialysis assays. Comparison of these in vitro cytotoxicity results with those obtained by an in vivo procedure (the Draize test) showed that the protein in the culture medium was essential for the establishment of good correlations. Thus, this in vitro test can be considered a reliable alternative in the assessment of surfactant toxicity, although its applicability to other types of eye irritant remains to be determined.

Prediction of eye irritancy potential of surfactants by cytotoxicity tests in vitro on cultures of human skin fibroblasts and keratinocytes.

The cytotoxicity of surfactants was evaluated on cultures of human skin fibroblasts and keratinocytes in order to predict their eye irritancy potential taking into account both immediate cytotoxicity after 2 hr of incubation and delayed cytotoxicity 24 hr after such incubation. The immediate cytotoxicity ranking of the surfactants, evaluated by MTT or neutral red assay after 2 hr of exposure in minimum Eagle's medium (MEM) without or with 10% foetal calf serum (FCS), was identical for both cell types. Keratinocytes were less sensitive than fibroblasts to all surfactants apart from Tween; however, cytotoxicity ranking remained the same for both cell types. The addition of 10% FCS to MEM reduced the cytotoxicity of surfactants, without modifying the ranking. Comparison of immediate and delayed cytotoxicity enabled the reversibility of cellular functions to be assessed by calculation of cell recovery rate (CRR). CRR values were positively related to cytotoxicity ranking for all surfactants except Brij. No correlation was found between EC(50) values of immediate or delayed cytotoxicity, under various experimental conditions, and ocular irritation scores in vivo. Brij surfactants, reported not to be irritant in vivo, showed a high degree of toxicity in the cell culture systems; however, a good correlation was found when analysis was carried out excluding these surfactants. In contrast, there was excellent correlation between CRR and ocular irritation scores in vivo, for all surfactants tested, including Brij.

Free-radical damage by UV or hypoxanthine-xanthine oxidase in cultured human skin fibroblasts: Protective effects of two human plasma fractions.

Two in vitro methods to investigate free radical damage to cultured human skin fibroblasts have been used: irradiation with UVA or UVB, producing intracellular free radicals and DNA damage, or free radical production by the enzymatic system hypoxanthine-xanthine oxidase, releasing free radicals into the culture medium. These methods differ not only in the location of the free radicals generated but also in their nature and kinetics. The antioxidant properties of two human plasma extracts A and B (derived from Cohn's fraction IV and Cohn's fraction I + III) were investigated before, during and/or after the oxidative stress. Protection was observed when the fractions were added concomitantly with the enzymatic system (at 5 g/litre, fractions A and B exhibited, respectively, 77 and 50% activity) or during UVB irradiation (37 and 68% activity for fractions A and B, respectively at 5 g/litre). A small degree of protection was observed against UVA damage. No preventive or restorative effect was observed with the UVB system. The two fractions prevented UVA damage (at 2.5 g/litre, fraction A and B elicited 22 and 23% activity, respectively) but only fraction A also exhibited a restorative effect (at 5 g/litre, activity was 26%). One of the protective mechanisms could be the enhancement of intracellular antioxidant enzyme activity by incubation of cells with fractions A and B (after 24 hr of contact with fraction B, total glutathione peroxidase and glutathione peroxidase Se-dependent activities were significantly increased by 60 and 42%, respectively, compared with control values).

Effect of biophysical changes on propidium iodide access to DNA during oxidative stress of cultured human skin cells.

The sensitive single-cell analytical techniques of flow cytometry and propidium iodide-binding have been used to examine the molecular effect of oxidative stress on cultured human skin fibroblasts. Cells synchronized by limited time attachment were exposed to a hypoxanthine-xanthine oxidase (HX/XO) system at different intervals after subculture. The characteristic feature of the treated population was a variation of the amount of nuclear DNA propidium iodide (PI)-fluorescence staining. Increased fluorescence intensity was observed with a shift of the G1/G0 and G2/M peak, which is dependent on both cell cycle stage and treatment level. When scavenger molecules (catalase, silybin) were added to the oxidative reaction, the nuclear DNA histogram of HX/XO-treated cells was similar to that obtained from untreated cells. In parallel, UV absorbance studies in vitro have shown that PI is capable of binding extensively to DNA when isolated from HX/XO-exposed cells, compared with control cells or HX/XO-exposed cells in the presence of scavengers. These results indicate that free radicals are responsible for the increase in fluorescence intensity in the HX/XO-exposed cells. This change in DNA stainability would be due to an opening of the DNA strands in situ, leading to an unmasking of new PI-binding sites on DNA. The strand separation may facilitate access on the fluorochrome to DNA, thereby enhancing dye binding. This flow cytometric assay based on DNA biophysical changes caused by free radicals is a useful means of measuring pro- and/or anti-oxidant potential.

Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol.

A three-dimensional culture of human keratinocytes exposed at the air-liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, (35)S-methionine incorporation, IL1-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the apical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD(50) values (40 J/cm(2)) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.

In vitro cytotoxic effects of enzymatically induced oxygen radicals in human fibroblasts: Experimental procedures and protection by radical scavengers.

Introduction of hypoxanthine and xanthine oxidase into human fibroblast cultures induces a dose-dependent cytotoxicity as a result of free-radical formation. The influence of medium, cell density and the power of recovery after free-radical attack were investigated. It appears that toxicity is higher in physiological Dulbecco phosphate buffer or Hanks' balanced salt solution than in modified Eagle medium, is inversely proportional to cell density and that damage is most often irreversible. Using this model, we studied the protective effects of a hydrosoluble flavonoid, silybin, and of a well known antioxidant, BHT (butylated hydroxytoluene). These molecules were administered before and during free-radical attack. With BHT significant protection was observed when it was added before free-radical attack (24% protection at a concentration of 10(-4)m) and before and during exposure (20% protection at a concentration of 10(-5)m). When silybin is applied during radical attack maximal activity is recorded at a concentration of 8 x 10(-4)m (45%), but the most interesting results are observed when 1 x 10(-4) and 8 x 10(-4)m are used, respectively, before and during radical exposure (63% of activity).

Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation.

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

Suicide by electrocution with low-voltage current.

Three cases of suicide by electrocution with low-voltage current were observed in five years (1994-1998) by medical clinical forensic examiners of an Emergency Forensic Unit of the Paris suburb among 2,000 external death examinations. The cases involved one woman, aged 72 and two men, aged 38 and 41. In the last two cases, electric burns were retrieved under bared electric wires, placed on the arms or fingers in order to realize a hand-to-hand electric circuit involving the heart muscle. In the other case, the electric circuit between mouth and foot also involved the heart muscle. Household low-voltage current delivered (220 V in France) had a sufficient strength to induce local muscular paralysis and heart fibrillation. In the three cases, blood samples taken have retrieved very high levels of muscular enzymes (CPK, LDH) correlated to the mechanism of electric death. The rareness of suicide by electrocution and its forensic characteristics are detailed in order to help the clinical forensic examiners, prosecutors, and police officers concerned by such death examinations.

Role of the appendageal pathway in the percutaneous absorption of pyridostigmine bromide in various vehicles.

We studied the percutaneous absorption of [14C]-labelled pyridostigmine bromide mixed into various vehicles through normal and appendage-free scar rat skin, in vitro during 72 h. At the end of the experiment, the percentages of the drug absorbed were higher for nerol 8% in ethanol (respectively 78.4 +/- 3.6% and 72.8 +/- 4.5% on normal and scar skin) and azone 5% in ethanol-propylene glycol (90:10) (respectively 76.4 +/- 4.4% and 57.2 +/- 7.1% on normal and scar skin). Propylene glycol 10% in ethanol inhibits pyridostigmine absorption: 9.9 +/- 2.6% and 2.2 +/- 1.2% vs 14.7 +/- 3.8% and 5.5 +/- 5.1% with ethanol on control and scar skin. The transappendageal pathway seems to be less important for nerol (55% to 82% of the absorption routes between 4 h and 32 h) and azone (60% to 79% of the absorption routes until 32 h) than for propylene glycol (63% to 96% of the absorption pathways during the whole experiment), dimethylsulfoxide (about 78% during the first 32 h) and ethanol (more than 50% during most of the time). These results show that it is possible to increase or decrease the percutaneous absorption, as well as to modulate the relative importance of the transepidermal route and the transfollicular pathway.

Percutaneous absorption of 5-iodo-2' deoxycytidine in the hairless rat and in man.

A study of percutaneous absorption of the anti-herpetic agent 5-iodo-2' deoxycytidine (IDC) was performed in vitro on the rat and on man and in vivo on the rat, using samples of normal skin and skin stripped of its stratum corneum. With normal skin, absorption was 1.84 cm.h1 10(-4) for the rat and 0.33 cm.h1 10(-4) for human skin; when the stratum corneum was absent, absorption was approximately 15 times higher. The results are discussed in terms of efficacy and toxicity of the drug.

Carbon-monoxide poisoning in young drug addicts due to indoor use of a gasoline-powered generator.

We report six fatal cases of unintentional carbon-monoxide poisoning which occurred in a house occupied by young people. The source of carbon monoxide was a gasoline-powered generator. For all victims, an external body examination was carried out and blood and urine samples collected. Blood carboxyhaemoglobin (COHb) was performed using an automated visible spectrophotometric analysis. Blood-alcohol level quantification was performed using gas chromatography and drug screening in urine was performed by a one-step manual qualitative immunochromatography (Syva Rapid test, Behring Diagnostics Inc.) for benzoylecgonine (the main metabolite of cocaine in urine), morphine, 11-nor-Delta(9)-THC-9-COOH (cannabinoids) and d-methamphetamine. In all victims the COHb value was as high or higher than 65%. No alcohol was found in blood samples, but urine samples were positive for methamphetamine, cocaine and cannabis in five cases and for opiates in one case. In four victims, the urine sample was positive for at least three drugs. The availability and accuracy of rapid toxicological screening is an important tool for the medical examiner at the immediate scene of a clinical forensic examination.