Benzidine mechanistic data and risk assessment: species- and organ-specific metabolic activation

The aromatic amine benzidine (BZ) has produced various tumors, including liver tumors, in mice, rats and hamsters. BZ forms DNA adducts in rodent liver, and it is positive in most genotoxicity tests. Only bladder tumors are produced in dogs and in humans who have been occupationally exposed, possibly related to the slow rate of liver detoxification by acetylation, allowing activation of BZ or its metabolites in urine. Despite these differences, risk assessment for humans, based on liver tumors in mice, was approximately predictive of the incidence of bladder tumors observed in industrially exposed humans.     

Prediction of minimal erythema dose with a reflectance melanin meter

The relationship between skin pigmentation and sensitivity to ultraviolet (UV) radiation-induced erythema was investigated in 60 healthy subjects of sun-reactive skin types I-V. Using a portable reflectance spectrometer, skin pigmentation was measured as the 'melanin index' (MI) in all subjects. A solar-simulated array of filtered UVA and UVB-emitting fluorescent lamps was then used to determine the UVB minimal erythema dose (MED) of each subject. MI readings and MED testing were both performed on the subjects' mid to upper backs. Using this technique, we found a close correlation between MI and MED. Comparison of the mean MI or MED of subjects with different skin types revealed progressive differences in MI and MED between all five skin types. Erythema dose-response curves, which provide further information about UV sensitivity, were also calculated for 43 subjects. A significant negative correlation was found between the gradients of these curves and both MI and MED, indicating that paler subjects respond more strongly to increments of UV above the MED than subjects with greater pigmentation. Our results indicate that although traditional, subjective means of predicting UV sensitivity to erythema are not without some value, MED correlates particularly strongly with objective measures of skin pigmentation. We therefore conclude that the reflectance spectrometer can rapidly and accurately predict UVB sensitivity, and should prove clinically useful for planning and optimizing UVB phototherapy.     

Pharmacokinetics and effect of food on the bioavailability of orally administered venlafaxine

Venlafaxine is a unique antidepressant currently under evaluation for treatment of various affective disorders. The pharmacokinetics and relative bioavailability of venlafaxine were evaluated in healthy volunteers after oral administration. The bioavailability of 50 mg of venlafaxine as a tablet relative to a solution was determined in a two-period randomized crossover study. The rate of absorption from the gastrointestinal tract was assessed by the time to peak plasma concentration (tmax), a model-dependent calculation of the first-order absorption rate constant, and a model-independent calculation of mean residence time. The extent of absorption was assessed by peak plasma concentration (Cmax) and area under the concentration-time curve (AUC). No statistically significant differences were observed between the two formulations for either the rate or extent of absorption. Similarly, systemic concentrations of the active O-demethylated metabolite did not significantly differ after administration of the two venlafaxine formulations. AUC ratios indicated that the relative bioavailabilities of the parent drug, and formulation of metabolite were approximately 98% and 92%, respectively, for the tablet versus the solution. A separate study was conducted to examine the influence of food on venlafaxine absorption from the 50-mg tablet. A standard, medium-fat breakfast eaten immediately before drug administration delayed the tmax of venlafaxine but did not affect Cmax or AUC. Therefore the tablet formulation of venlafaxine is bioequivalent to the oral solution, and the presence of food appears to decrease the rate but not the extent of absorption of venlafaxine from the tablet formulation.     

Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42

We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTPgammaS to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTPgammaS appeared to act via a small G-protein, since it was active in lysates ofD. discoideum mutants missing either the alpha2- or beta-subunit of the heterotrimeric G-protein required for chemoattractant-induced actin polymerization in living cells. Furthermore, recombinant Cdc42, but not Rho or Rac, induced polymerization in the cell-free system. The Cdc42-induced increase in filamentous actin required GTPgammaS binding and was inhibited by a fragment of the enzyme PAK1 that binds Cdc42. In a high speed supernatant, GTPgammaS alone was ineffective, but GTPgammaS-loaded Cdc42 induced actin polymerization, suggesting that the response was limited by guanine nucleotide exchange. Stimulating exchange by chelating magnesium, by adding acidic phospholipids, or by adding the exchange factors Cdc24 or Dbl restored the ability of GTPgammaS to induce polymerization. The stimulation of actin polymerization did not correlate with PIP2 synthesis.     

Gonadotropin-releasing hormone immunoreactivity in the nasal epithelia of adults with Kallmann's syndrome and isolated hypogonadotropic hypogonadism and in the early midtrimester human fetus

GnRH-secreting neurons are known to originate in the epithelium of the medial olfactory placode, whence they migrate along the axons of the terminal nerve via the forebrain and into the hypothalamus. Synaptic contact between the developing olfactory bulbs and fascicles of the vomeronasal, terminal, and olfactory nerves does not occur in Kallmann's syndrome. Consequently, there is migration arrest of GnRH cells and partial or complete failure of formation of the olfactory bulbs, resulting in severe olfactory deficit and hypogonadotropic hypogonadism. In the present study, using an immunofluorescent, double immunostaining technique and confocal laser scanning microscopy, we observed GnRH-immunoreactive neurons in the hypothalamus of a 14-week-old human fetus. However, migration of GnRH neurons was not complete, and indeed, such cells were seen to be migrating along terminal nerve fascicles beneath the cribriform plate in a 16-week-old fetus. The same immunofluorescent technique demonstrated the presence of GnRH cells in biopsies of nasal mucosa obtained from three adults with Kallmann's syndrome, one normosmic subject with hypogonadotropic hypogonadism, and a eugonadal male cadaver. These findings are consistent with two different interpretations: the nasal GnRH neurons may be vestigial, representing cells that failed to migrate during embryogenesis; alternatively, they may have been generated de novo later in life, a possibility consistent with the recognized plasticity of human postnatal olfactory neuroepithelium. They also reveal that subjects with the normosmic (i.e. non-Kallmann's) form of GnRH deficiency are able to synthesize immunologically recognizable GnRH, implying that failure of GnRH synthesis is not responsible for this type of hypogonadotropic hypogonadism.     

The role of the genome project in determining gene function: insights from model organisms

Peptido-leukotrienes are short-lived organic molecules known to have potent biological effects as mediators of inflammation, hypersensitivity and respiratory disorders. However, little is known concerning their effects on bone cells. We have shown previously that stromal cells isolated from a human giant cell tumor secrete 5-HETE (5-hydroxyeicosatetraenoic acid) and the peptido-leukotrienes, also known as the cysteinyl leukotrienes LTC4, LTD4, and LTE4. These eicosanoids were shown to stimulate the multinucleated giant cells obtained from these tumors to form resorption lacunae on sperm whale dentine. Here, we show that the peptido-leukotrienes also stimulate isolated avian osteoclast-like cells to form resorption lacunae and to increase their content of tartrate-resistant acid phosphatase. LTD4 increased 45Ca release from murine calvarial bone organ cultures, but not from fetal rat long bone cultures. Isolated avian osteoclast-like cells were chosen to perform receptor binding studies, as this population is the most homogeneous source of osteoclasts available. After the precursors had fused to form multinucleated cells, receptor binding assays were performed. Scatchard analysis of saturation binding data showed a single class of binding sites, with a dissociation constant (Kd) of 0.53 nM and a receptor density of 5,200 receptors per cell. Competition binding studies showed receptor specificity using a specific LTD4 receptor antagonist ZM 198,615. These data show that the peptido-leukotrienes activate highly enriched populations of isolated avian osteoclast-like cells, and also that specific LTD4 receptors are present in this cell population.     

Evidence for muscarinic 3 receptor mediated ion transport in HT29 cells studied by X-ray microanalysis

Changes in elemental content in response to muscarinic drugs in HT29 cells were investigated by X-ray microanalysis. Acetylcholine (ACh) and carbachol (Cch), both agonists binding to muscarinic receptors, induced a decrease of the intracellular Cl and K content. This agrees with the notion that these agonists induce electrolyte and water secretion. Atropine, a non-selective antagonist of muscarinic receptors, inhibited the decrease in K and Cl caused by ACh and Cch, and instead caused an increase of the Cl and K concentrations. A similar inhibition was found in the case of the selective muscarinic 3 receptor antagonist P-F-HHSiD. In contrast, the selective muscarinic 2 receptor antagonist AF-DX 116 did not inhibit Cch-activated secretion of K and Cl. A slight inhibition of ACh induced ion secretion was seen, but this inhibition was weak compared to that caused by P-F-HHSiD. Treatment with U-73122, an inhibitor of phospholipase C, blocked ACh or Cch induced ion secretion. These results suggest that ACh and Cch stimulated secretion of Cl and K is mediated by muscarinic 3 receptors via the inositol-1,4,5-trisphosphate (IP3) dependent pathway.