Human percutaneous absorption of a direct hair dye comparing in vitro and in vivo results: Implications for safety assessment and animal testing

Although in vitro skin absorption studies often detect small residues of applied test material in the epidermis/dermis, it is uncertain whether the residue is within the living skin. We studied the dermal absorption of a hair dye hydroxyanthraquinone–aminopropyl methyl morpholinium methosulphate (HAM) in human skin in vivo and in vitro. In vivo, skin (back and scalp) received 0.5% HAM in a commercial formulation at 20 μg/cm² After 0.5 or 48 h, skin was tape stripped, followed by cyanoacrylate biopsies (CAB). Sebum from scalp sites was collected for 48 h. In vitro, skin was treated with 20 mg/cm² dye for 0.5 h, penetration determined after 24 h. In vivo, at 0.5 h, total recovery (back) was 0.67 μg/cm² (tape strips + CAB). Fluorescence microscopy showed HAM in the hair follicle openings (HFO). At 0.5 h, scalp tape strips contained 1.80 μg/cm², HFO 0.82 μg/cm². At 48 h, HFO contained 0.21 μg/cm², sebum 0.80 μg/cm². In vivo, skin residues were in the non-living skin and eliminated via desquamation and sebum secretion. In vitro, the SC contained 1.50 μg/cm², epidermis/dermis 0.86 μg/cm², receptor fluid < 0.04 μg/cm², a total of 0.90 μg/cm² was considered to be bioavailable. In vitro epidermis/dermis residues were nearly identical to those located in non-living skin in vivo. In conclusion, in vitro percutaneous penetration studies may produce seemingly bioavailable material , which raises the need for a Threshold of Skin Absorption (TSA) addressing a negligible dermal absorption in order to avoid unnecessary in vivo toxicity studies on substances that produce no significant human systemic exposure.     

Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentration

The cytotoxicity of 10 chemicals from the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) list (nos 21–30) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the MEIC programme organized by the Scandinavian Society of Cell Toxicology. The MTT test was used as the endpoint of cytotoxicity after 24hr of exposure to the chemicals. Theophylline, phenobarbital and paraquat were the least cytotoxic compounds in the cellular systems (IC₅₀ = 450-17,000 μm) except for the 3T3 cells. The seven remaining chemicals (dextropropoxyphene, propranolol, arsenic trioxide, cupric sulfate, mercuric chloride, thioridazine and thallium sulfate) showed a similar relative cytotoxic ranking in the four in vitro systems in the lower range of concentrations (IC₅₀ = 2–350 μm). The data suggest that these 10 chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or species specific. The correlation between in vitro data and human lethal blood concentrations showed that the predictability of the in vitro systems was similar to that of in vivo rodent tests (LD₅₀) only when low cytotoxic concentrations (IC₁₀) were used for correlation.     

In vivo and in vitro microdialysis sampling of free fatty acids

Microdialysis is a technique that allows continuous sampling of compounds from the interstitial fluid of different tissues with minimal influence on surrounding tissues and/or whole body function. In the present study, the feasibility of using microdialysis as a technique to sample free fatty acids (FFA) was investigated both in vitro and in vivo, by use of a high molecular weight (MW) cut-off membrane (3 MDa) and a push–pull system to avoid loss of perfusion fluid through the dialysis membrane. The relative recovery was examined in vitro for three different concentrations of radiolabelled oleic acid-BSA solutions (oleic acid:BSA molar ratio 1:1) and for various temperatures and flow rates. The recovery of oleic acid was found to be dependent on the concentration of analyte in the medium surrounding the membrane (17.3%, 29.0% and 30.6% for 50, 100 and 200 μM oleic acid-BSA solutions, respectively). Addition of 0.25% BSA to the perfusion fluid resulted, however, in a concentration-independent recovery of 31.4%, 28.1% and 28.1% for the 50, 100 and 200 μM solutions, respectively.

The capability of the method to measure FFA together with glycerol was investigated in vivo in visceral adipose tissue of rats, before and after lipolytic treatment with the β₃-adrenergic agent, BRL37344. BRL37344 caused an increase in both dialysate FFA and glycerol, although the increase was markedly higher for glycerol, amounting to 24.5% and 329.2% increase from baseline, respectively. Subsequent in vitro test of probe performance revealed a decrease in the dialysing properties with regard to FFA, but not glycerol. This suggests that clogging of the membrane pores after 110 min prevented the measurement of the full FFA response in vivo.     

The effect of the venom of the yellow Iranian scorpion Odontobuthus doriae on skeletal muscle preparations in vitro

The yellow Iranian scorpion Odontobuthus doriae can cause fatal envenoming, but its mechanism of action is unclear. One of the reported manifestations of envenoming is moderate to severe involuntary tremor of skeletal muscle. In order to understand better the mechanism of action of this venom on skeletal muscle function, we examined the effects of the venom in vitro on chick biventer cervicis (CBC) and mouse hemidiaphragm (MHD) nerve muscle preparations. O. doriae venom (0.3–10 μg/ml) initially increased and then decreased twitch height. The venom also caused contracture in both preparations. In mouse triangularis sterni preparations, used for all intracellular recording techniques, the venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials and endplate potentials in response to single-shock stimulation. With extracellular recording techniques, scorpion venom (1 μg/ml) was found to cause changes to the perineural waveform associated with nerve terminal action potentials consistent with effects on Na⁺ and K⁺ currents. The main facilitatory effects of O. doriae venom are likely to be due to toxins that affect Na⁺ channels in nerve–muscle preparations similar to most Old World scorpion venoms, but blocking effects on K⁺ channels are also possible. Such effects could lead to initial enhancement of transmitter release that could underlie the muscle tremors seen in victims. Toxins acting on Na⁺ and K+ currents have been isolated from the venom [Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E., Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat, J., 2005. OD1, the first toxin isolated from the venom of the scorpion Odontobuthus doriae active on voltage-gated Na⁺ channels. FEBS Lett. 579, 4181–4186; Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian scorpion Odontobuthus doriae. FEBS Lett. 580, 6254–6258]; however, the muscle paralysis seen at higher concentrations of venom may be due to additional, as yet uncharacterised, components of the venom.     

Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae)

Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10 μg/ml for UD, 6 μM for celecoxib and 42 μM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.     

In vitro biological efficiency of tenocyclidine – TCP and its adamantane derivative TAMORF

Tenocyclidine – TCP showing a broad spectrum of pharmacological activity including antidotal effect in organophosphorus compounds poisoning, radioprotective and anticancer effects. We investigated in vitro interactions of TCP and its adamantane derivative – TAMORF with human erythrocyte acetylcholinesterase (AChE). Moreover, their genotoxicity and radioprotective activity on human white blood cells were studied using the alkaline comet assay, viability testing and the analysis of the structural chromosome aberrations. The tested compounds were found to be weak inhibitors of AChE, for TCP IC₅₀ = 1 × 10⁻⁵ M and for TAMORF IC₅₀ > 1 × 10⁻³ M, without reactivating and protective effects on AChE inhibited by soman. Results suggest that TCP modified by the replacement of the cyclohexyl ring with an adamantly ring and piperidine with morpholine group (TAMORF) have lower toxicity. Both compounds possess low cytotoxicity and radioprotective activity, but TAMORF also shows cell growth inhibitory effects. To clarify differences in their biological efficiency observed in vitro and in vivo, additional analyses are necessary. Since TAMORF was found to significantly inhibit cell growth and proliferation in vitro, it is reasonably to consider it as a source molecule promising for further modifications and development of more potent substances with antitumor properties rather then radioprotector or antidote in organophosphorus poisoning.     

Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl₂ and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl₂, CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25–10 μM-Cd. Cells exposed to CdCl₂ accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl₂ the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 μM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 μM, therefore, the difference in Cd accumulation between CdCl₂ and CdMt was greater (5–6 times) than at concentrations below 2 μM (1–2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 μM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl₂ was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1–2 μM) to CdCl₂ intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10–20 μM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20–35 μM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 μM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl₂ to cell cultures in a serum-free medium. Whereas CdCl₂ in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 μM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl₂ is associated with a difference in Cd distribution.     

Lyophilized Cheliensisin A submicron emulsion for intravenous injection: characterization, in vitro and in vivo antitumor effect

Growing attentions have been focused on natural antitumor drugs. Recently, a novel and potent antitumor drug Cheliensisin A (GC-51) with broad-spectrum efficiency has been developed. However, due to its poor water solubility and chemical instability, choosing the appropriate dosage form is of great significance. This study aimed at developing a lyophilized submicron emulsion for GC-51 and further improving the therapeutic index of the drug. The resultant lyophilized GC-51 submicron emulsion was much more stable than its solution, which can be stored for years without significant change on physicochemical properties. And its solubility was increased from 6.74 ± 0.14 to 2.00 ± 0.10 mg mL⁻¹. The 50% inhibitory concentration IC₅₀ values were calculated from growth curves by MTT assay on various tumor cell lines. Compared with the IC₅₀ of GC-51 crude drug, that of lyophilized GC-51 submicron emulsion decreased from 24.04 ± 1.97 to 8.23 ± 1.84 μg mL⁻¹ on HepG2, and from 31.08 ± 2.56 to 10.85 ± 2.09 μg mL⁻¹ on CT-26, from 17.90 ± 1.83 to 7.49 ± 1.87 μg mL⁻¹ on HeLa and from 16.38 ± 2.41 to 10.13 ± 2.12 μg mL⁻¹ on A549, respectively. In the time-dependent assay of tumor cell viability, lyophilized GC-51 submicron emulsion exhibited significantly lower inhibition rate in the initial action times, but increased gradually afterwards. That means lyophilized submicron emulsion as the vector for GC-51 had some protective and delayed release effect. Further, the in vivo therapeutic efficacy was measured in pulmonary metastasis of colon cancer-bearing BALB/c mice model. An obvious enhanced antitumor activity was observed after administration of lyophilized GC-51 submicron emulsion (P < 0.05), which increased from 22.78 ± 3.5 to 41.42 ± 4.2% compared with GC-51 injection. And the life span of tumor-bearing mice in lyophilized GC-51 submicron emulsion group was significantly longer than that of the mice in GC-51 injection and normal saline groups. Compared with crude drug, the lyophilized GC-51 submicron emulsion showed a significantly higher antitumor efficiency both in vivo and in vitro, suggesting a potential application in tumor chemotherapy.     

Ordered mesoporous silica induces pH-independent supersaturation of the basic low solubility compound itraconazole resulting in enhanced transepithelial transport

The majority of innovative drug candidates are poorly water soluble and exhibit basic properties. This makes them highly dependent on the in vivo encountered acid–neutral pH sequence to achieve a sufficient dissolution and thus absorption. In this study, we evaluated the pH-independent generation of intraluminally induced supersaturation of the model compound itraconazole and its beneficial effect on the extent of absorption in the Caco-2 system and the rat in situ perfusion system. Local supersaturation was obtained by means of a solvent shift method and a novel formulation strategy based on ordered mesoporous silica (OMS) as a carrier. In vitro results evidenced that both methods were capable of creating a supersaturated state of itraconazole in fasted state simulated intestinal fluid (FaSSIF) when no preceding acidic dissolution was simulated. The extent of supersaturation exceeded 21.9 and 9.6 during at least 4 h for the solvent shift method and OMS as a carrier, respectively. As compared to saturation conditions (0.09 ± 0.01 μg), supersaturation induced by the solvent shift method as well as by the use of OMS increased transport across a Caco-2 cell monolayer more than 16-fold, resulting in the basolateral appearance of 2.20 ± 0.29 μg and 1.46 ± 0.03 μg itraconazole after 90 min, respectively. In the absence of an acid–neutral pH sequence, the performance of the marketed product Sporanox^® was inferior with total transport amounting to 0.12 ± 0.03 μg after 90 min. Enhanced absorption was confirmed in the in situ perfusion model where OMS was able to boost total transport of itraconazole after 60 min from 0.03 ± 0.01 nmol cm⁻¹ to 0.70 ± 0.09 nmol cm⁻¹ compared to saturated equilibrium conditions in FaSSIF. The solid dosage form Sporanox^® again failed to achieve a similar extent of absorption enhancement (0.29 ± 0.01 nmol cm⁻¹). These findings suggest that intraluminal supersaturation can be created by the use of OMS and that preceding dissolution of basic compounds in the acidic medium of the stomach is not required to allow for efficient intestinal absorption. The use of OMS appears to be a promising strategy for the delivery of especially basic low solubility compounds in patients suffering from hypochlorhydria; the pH independency may also result in a more reproducible systemic exposure.     

Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca transport proteins and causing dysregulation of Ca homeostasis

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca²⁺ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca²⁺ within these cells in the 5–60 μM concentration range (EC₅₀, 21 μM). TBBPA also causes cell death (LC₅₀, 18 μM) partly via apoptosis, involving Ca²⁺-dependent mitochondrial depolarisation. Studies on intracellular Ca²⁺ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA) at low concentrations (IC₅₀, 0.4 to 1.2 μM) and also activate the Ryanodine receptor Ca²⁺ channel within the 0.4–4 μM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca²⁺ signalling, by directly affecting Ca²⁺ transport proteins.     

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and H-deoxyglucose-6-phosphate release from human red blood cells

, and . Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and ³H-deoxyglucose-6-phosphate release from human red blood cells. Toxicon 27, 1297–1305, 1989.—At a low concentration of Naja naja kaouthia cardiotoxin (3 μM) Ca²⁺, Sr²⁺ and Ba²⁺ (2 mM), had little to no effect on ³H-deoxyglucose-6-phosphate (³H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (≥ 10 μM), Ca²⁺ (2 mM), but not Sr²⁺ or Ba²⁺, significantly enhanced ³H-dGlu-6-p and hemoglobin release. Mn²⁺ (2 mM) almost completely inhibited ³H-dGlu-6-p release and hemolysis at both the 3 μM and 10 μM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 μM), Ca²⁺ at low concentrations (0.5 mM) enhanced ³H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 μM) induced ³H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. ³H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between ³H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca²⁺-dependent. The Ca²⁺-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A₂ contamination in the toxin fraction. Mn²⁺ is an effective antagonist of cardiotoxin action.     

In vitro dermal absorption of pesticides: VI. In vivo and in vitro comparison of the organochlorine insecticide DDT in rat, guinea pig, pig, human and tissue-cultured skin

In vitro dermal absorption tests were conducted with the ¹⁴C-ring-labelled pesticide, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) dissolved in acetone and applied to dermatomed skin (0.5 mm) of a number of species at dose rates of 16–27 μg/cm². Skin absorption was determined for 48 hr after exposure using in vitro flow-through cells. Skin absorption was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution added to the percentage recovery for the methanol washes of the skin at 48 hr and the skin digest samples. Two receiver solutions, Ringer's saline (used with Moody aluminium cells), and Hanks' HEPES buffered saline with 4% serum albumin (used with Bronaugh flow-through cells) were used. Listed in decreasing order, the total percentage in vitro dermal absorptions obtained by 48 hr after exposure for the five skin types were: 42 ± 2.6% [hairless guinea pig; Hanks' receiver (HR)], 34 ± 10.5% (rat; HR), 28 ± 13.2% [Testskin; Ringer's receiver (RR)], 28 ± 2.9% (human; HR), 22 ± 3.3% (Testskin; HR), 18 ± 6.2% (pig; RR) and 14 ± 2.1% (pig; HR). The percentage ¹⁴C activity recovered in soapy water rinses of the skin specimens at 24 hr, and for methanol skin washes and skin digests at 48 hr, and of ¹⁴C-labelled volatiles collected in air traps are reported. Data obtained with pig and Testskin for DDT using the Moody flow-through permeation cell was compared with that obtained using the Bronaugh cell. Significantly greater (P < 0.05) percentage recovery was obtained for the soap washes at 24 hr of the skin following the Bronaugh procedure than was obtained with the Moody method. Comparative in vivo studies demonstrated urinary recovery was 2 ± 0.5 and 15 ± 1.7% for rats (dose rate; 6 μg/cm²) and guinea pigs (dose rate: 9 μg/cm²), respectively. Total faecal recovery was 20 ± 1.9 and 44 ± 2.75% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after dosing demonstrated total tissue recovery of 51 ± 5.6% in rats but of only 3 ± 0.7% in guinea pigs. Including the ¹⁴C activity extracted from the skin removed from the dose site at 14 days after exposure, the total recovery of dermally absorbed residues was 73 ± 5.9 and 62 ± 4.1% for rats and guinea pigs, respectively. Recovery of ¹⁴C from soapy water skin washes conducted at 24 hr after exposure was 3 ± 1.4 and 14 ± 1.8% for rats and guinea pigs, respectively, and this was significantly less than that obtained by both the Bronaugh and Moody in vitro procedures. Skin patch recovery was 24% for both rats and guinea pigs. In summary, the in vitro data underestimed the degree of dermal absorption observed in vivo for both rats and guinea pigs, and this was thought to be due to an overly vigorous removal of the pesticide skin deposit by the soap washing procedures used in vitro in comparison with the in vivo washing procedure.     

In vitro dermal absorption of pesticides: IV. In vivo and in vitro comparison with the organophosphorus insecticide diazinon in rat, guinea pig, pig, human and tissue-cultured skin

In vitro skin absorption tests are currently being developed as an alternative to in vivo animal tests for predicting the degree of occupational exposure to pesticides. In the study reported here, in vitro percutaneous absorption tests were conducted with the ¹⁴C-ring-labelled pesticide, diazinon, dissolved in acetone and applied to the dermatomed skin (0.5 mm) of a number of species at a dose rate of 9.5–16.7 μg/cm². Skin permeation was determined for 48 hr after exposure using an in vitro flow-through system. Skin permeation was calculated from the sum of the percentage recovery of ¹⁴C activity in the receiver solution and the percentage recovery obtained in methanol washes of the skin at 48 hr and in skin digests. Listed in decreasing order, the total percentage in vitro dermal absorptions (mean ± SD) obtained by 48 hr after exposure for the five skin types were: 47 ± 3.4% (rat), 36 ± 0.9% (tissue cultured Testskin), 33 ± 2.8% (hairless guinea pig), 20 ± 3.1% (human) and 15 ± 13.1% (pig). The percentage recoveries in soapy water skin washes at 24 hr, in methanol washes and skin digests at 48 hr and of ¹⁴C-labelled volatiles collected in air traps at 48 hr after exposure are reported. Comparative in vivo studies demonstrated 37 ± 0.8 and 24 ± 5.7% recovery of ¹⁴C in the urine of rats (dose rate, 6 μg/cm²) and hairless guinea pigs (dose rate, 5 μg/cm²), respectively, by 14 days after exposure. Total faecal recovery 14 days after exposure was 18 ± 0.4 and 4 ± 0.9% for rats and guinea pigs, respectively. Analysis of tissue taken at autopsy 14 days after exposure demonstrated a total tissue recovery of 0.6 ± 0.1% [¹⁴C]diazinon in rats and 1 ± 0.2% in hairless guinea pigs. The total recovery in skin removed from the dose site at 14 days after exposure was 0.2 ± 0.02% and 0.1 ± 0.05% in rats and hairless guinea pigs, respectively. Recovery of radioactivity from soapy water skin washes conducted at 24 hr after exposure was 21 ± 3.8% for rats and 2 ± 0.1% for hairless guinea pigs. Recovery in skin patches was 23 ± 5.4% and 73 ± 2.9% in rats and hairless guinea pigs, respectively. The in vitro data for dermal absorption of [¹⁴C]diazinon for rats (47 ± 3.4%) and hairless guinea pigs (33 ± 2.8%) were in good agreement with the data observed for rats (56 ± 1.03%) and hairless guinea pigs (28 ± 6.0%) in vivo. This study supported the use of in vitro skin absorption tests as an alternative to in vivo animal testing.     

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H₂O₂/Cl⁻ system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H₂O₂/Cl⁻ system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC₅₀ = 1.1 ± 0.3 μM) is the most efficient inhibitor of the MPO/H₂O₂/Cl⁻ system and nimesulide (IC₅₀ = 2.1 ± 0.3 μM) is more active than the other NSAIDs of the oxicam family (IC₅₀ = 8–12 μM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H₂O₂/Cl⁻ system. Flufenamic acid has the best inhibiting activity towards the MPO/H₂O₂/Cl⁻ system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.     

Lung tissue binding of iodobenzyl-propanediamine: Involvement of beta-adrenergic receptors

The basic compound N-N-N′-trimethyl-N′-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propanediamine (HIPDM) accumulates in human and rabbit lungs, where it forms a slowly effluxable pool. In isolated perfused rat lung, HIPDM is taken up by a saturable, energy-independent mechanism, which is competitively inhibited by imipramine, chlorpromazine and propranolol. To ascertain whether beta-adrenergic receptors are involved in the binding process of HIPDM to lung tissue, the ability of unlabelled HIPDM to displace the beta-adrenergic receptor ligand [¹²⁵I]iodocyano-pindolol (ICYP) from rabbit lung beta-receptors was examined. Lung microsomal membrane fractions (75 μg ml⁻¹) were incubated at 37°C for 3 h with 68 pM ICYP (with or without 1 μM of (±)-propranolol) in the presence of HIPDM (10⁻¹⁰−10⁻³ M). Bound and free radioactivity were separated through glass-fibre filters and the retained radioactivity was counted in a gamma-spectrometer. HIPDM competed with ICYP for beta-adrenoceptors (13% displacement at 10⁻⁵ M, 50% at 5 × 10⁻⁵ M, and 90% at 2 × 10⁻⁴ M). The inhibition curve of ICYP binding by HIPDM was similar to that observed for (−)-noradrenaline. Although the results of the in vitro studies cannot be extrapolated to in vivo conditions, they suggest that beta-adrenergic receptors may be involved in the observed lung uptake of the basic amine HIPDM.     

Role of tachykinin NK3 receptors in the release and effects of nerve growth factor in human isolated bronchi

The nerve growth factor (NGF) is a neurotrophic factor essential for the development and survival of neurons. It has also been identified as a mediator of inflammation and can cause airway hyperresponsiveness [Frossard et al., Eur. J. Pharmacol. 500, 453 (2004)]. Evidence in rodents suggests a link between tachykinins, the sensory nerves, and NGF. Recent evidence shows that NGF is released by the proinflammatory cytokine interleukin-1β and induces hyperresponsiveness to the tachykinin NK1 receptor agonist [Sar⁹,Met(O₂)¹¹]SP in isolated human bronchi. The aim of this study was to determine the role of sensory nerves through the effect of the tachykinin NK3 receptor antagonist SR142801 in the interleukin-1β effects and/or the NGF-induced airway hyperresponsiveness. SR142801 (0.1 μM) abolished the interleukin-1β (10 ng/ml, 21 °C, 15 h)-induced increased NGF release from isolated human bronchi in vitro (P < 0.05). In organ bath studies, SR142801 also abolished the interleukin-1β-induced airway hyperresponsiveness to [Sar⁹,Met(O₂)¹¹]SP (0.1 μM) (P < 0.05). SR142801 also inhibited the NGF-induced airway hyperresponsiveness (P < 0.01). This study suggests tachykininergic sensory nerves to be involved in the interleukin-1β-induced NGF release and airway hyperresponsiveness.     

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

, , , and . Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon 27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.

Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.

The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

Citral, an inhibitor of retinoic acid synthesis, attenuates the frequency and severity of branchial arch abnormalities induced by triazole-derivative fluconazole in rat embryos cultured in vitro

The clinically used antimycotic fluconazole (fluco) is teratogenic in rodents. Exposure in vitro to fluco, other investigated azoles (triadimefon, triadimenol, flusilazole, ketoconazole and imazalil) or retinoic acid (RA), is correlated to branchial arch abnormalities. Inhibition of RA degradation has been suggested as the azole-related mechanism. Citral is a RA synthesis inhibitor. E9.5 rat embryos were cultured for 48 h in normal serum or exposed in vitro to fluco 125 μM, citral 200 μM or co-exposed to the two molecules to test the hypothesis that citral attenuates fluco-related teratogenic effects. Some embryos were cultured for 12 extra hours, and cranial nerves immunodetected. Fluco induced typical abnormalities, including branchial arch and cranial nerve defects. The co-exposure to fluco + citral was significantly effective in reducing branchial arch and cranial nerve defects, supporting the hypothesis that citral balances the fluco-induced RA concentration increase. However, other fluco-related effects were unalterated by citral.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.