The calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS blocks CGRP and adrenomedullin vasoactive responses in the microvasculature.

Calcitonin gene-related peptide (CGRP) is a potent microvascular dilator neuropeptide that is considered to play an essential role in neurogenic vasodilatation and in maintaining functional integrity in peripheral tissues. We have examined the effect of the nonpeptide CGRP antagonist BIBN4096BS on responses to CGRP and the structurally related peptide adrenomedullin, AM, in murine isolated aorta and mesentery preparations, and in the cutaneous microvasculature in vivo. We show for the first time that BIBN4096BS is an effective antagonist of CGRP and AM responses in the murine mesenteric and cutaneous microvasculature, and of CGRP in the murine aorta. After local administration, BIBN4096BS selectively inhibits the potentiation of microvascular permeability in the cutaneous microvasculature by CGRP and AM, with no effect on responses induced by other microvascular vasodilators. BIBN4096BS reversed both newly developed and established vasoactive responses induced by CGRP. The ability of CGRP to potentiate plasma extravasation was lost when coinjected with compound 48/80 (where mast cells would be activated to release proteases), but regained when soybean trypsin inhibitor was coinjected with compound 48/80. These results demonstrate that BIBN4096BS is a selective antagonist of responses induced by CGRP and AM in the mouse microvasculature, and CGRP in the mouse aorta. The ability of BIBN4096BS to block an established CGRP microvascular vasodilatation indicates that the sustained vasodilator activity of CGRP is due to the retention of the active intact peptide and the continued involvement of the CGRP receptor.

Antimutagenicity of ethanol extracts of bee glue against environmental mutagens.

The antimutagenicity of ethanol extracts of bee glue (propolis) (EEBG) was evaluated, using Salmonella typhimurium strain TA98 as a test model, against two direct mutagens, 4-nitro-O-phenylenediamine (4-NO) and 1-nitropyrene (1-NP), and two indirect mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo[a]pyrene (B[a]P) with S9 mix. EEBG was shown to suppress the mutagenicity of these compounds in a dose-dependent fashion. To delineate the mechanism of action of the antimutagenic effects of EEBG on the two indirect mutagens IQ and B[a]P, two possible points of blocking were considered: (1) cytochrome P-450 activity (route 1) and (2) interaction with microsome-generated proximate mutagens to generate an inactive complex (route 2). Our results clearly demonstrated, at a very low dose, remarkable suppression of the mutagenicity of both compounds by inhibiting either route 1 or route 2 pathway. Further studies indicated that EEBG was capable of inhibiting both the activities of hepatic cytochrome P-450 IA1-linked 7-ethoxyresorufin-O-deethylase (EROD) and IA 2-linked 7-ethoxycoumarin-O-deethylase (ECD) in a similar dose-dependent manner. Taken together, we demonstrated that EEBG was a good inhibitor for mutagenicity of direct mutagens, 1-NP and 4-NO, as well as for the indirect mutagens IQ and B[a]P in the presence of S9 mix via inactivation of microsomal enzyme activities (e.g. EROD and ECD) or antagonizing metabolic generation of the proximate mutagens of IQ and B[a]P.

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage.

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.

Ascorbic acid inhibits lipid peroxidation but enhances DNA damage in rat liver nuclei incubated with iron ions.

In this report we studied DNA damage and lipid peroxidation in rat liver nuclei incubated with iron ions for up to 2 hrs in order to examine whether nuclear DNA damage was dependent on membrane lipid peroxidation. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) and DNA damage was measured as 8-OH-deoxyguanosine (8-OH-dG). We showed that Fe(II) induced nuclear lipid peroxidation dose-dependently but only the highest concentration (1.0 mM) used induced appreciable 8-OH-dG. Fe(III) up to 1 mM induced minimal lipid peroxidation and negligible amounts of 8-OH-dG. Ascorbic acid enhanced Fe(II)-induced lipid peroxidation at a ratio to Fe(II) of 1:1 but strongly inhibited peroxidation at ratios of 2.5:1 and 5:1. By contrast, ascorbate markedly enhanced DNA damage at all ratios tested and in a concentration-dependent manner. The nuclear DNA damage induced by 1 mM FeSO4/5 mM ascorbic acid was largely inhibited by iron chelators and by dimethylsulphoxide and mannitol, indicating the involvement of OH. Hydrogen peroxide and superoxide anions were also involved, as DNA damage was partially inhibited by catalase and, to a lesser extent, by superoxide dismutase. The chain-breaking antioxidants butylated hydroxytoluene and diphenylamine (an alkoxyl radical scavenger) did not inhibit DNA damage. Hence, this study demonstrated that ascorbic acid enhanced Fe(II)-induced DNA base modification which was not dependent on lipid peroxidation in rat liver nuclei.

Hemolytic effects of dehydroepiandrosterone in vitro.

Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland which decreases with age after adolescence, is available as a over-the-counter product. This study demonstrates that DHEA induced lysis of human red blood cells (RBCs) in a concentration-dependent manner, with ca. 70% hemolysis at 2 mM DHEA at 37 degrees C for 1 hr. Hemolysis induced by 2 mM DHEA was rapid and involved neither hemoglobin oxidation nor lipid peroxidation. The hemolysis was also not inhibited by addition of EDTA, catalase, superoxide dismutase, glucose or a radical scavenger including mannitol, dimethylsulfoxide and alpha-tocopherol, indicating a non-oxidative mechanism. RBCs stored overnight before incubation with DHEA were hemolyzed to a lesser extent than the freshly prepared RBCs. Light microscopy of the fresh RBCs following 1-h incubation with 2 mM DHEA revealed thickened and cup-shaped deformity of the membranes, suggesting a change in the membrane structure possibly due to the intercalation of the steroid into the membranes.

UVA-potentiated damage to calf thymus DNA by Fenton reaction system and protection by para-aminobenzoic acid.

Calf thymus DNA was irradiated with low-intensity UVA (main output at 365 nm, 2 mW cm-2 or 36 kJ m-2 for 30 min), and the role of metal ions, hydrogen peroxide and reactive oxygen species (ROS) was examined. DNA damage was measured as thiobarbituric acid-reactive substances (possibly from degradation of deoxyribose) and as changes in ethidium bromide-DNA fluorescence due to unwinding from strand breaks. Under the present experimental conditions, UVA alone or in the presence of H2O2 had no effect on DNA but slightly enhanced the damage by iron/EDTA. Ultraviolet A strongly enhanced DNA damage (ca four- to five-fold) by the Fenton reaction system (50 microM Fe2+/100 microM EDTA + 0.5 mM H2O2). The results suggest that the Fenton reaction system was "photosensitized" to damage DNA by low-intensity UVA radiation. The enhanced damage by UVA was attributed in part to the reduction of Fe3+ to Fe2+. Ultraviolet A had no effect when iron (ferric or ferrous) ions were replaced by Cu2+, Zn2+, Mn2+ or Cd2+. The ROS involved in the UVA-enhanced damage to DNA by the Fenton reagents were OH and, to a lesser extent, superoxide anions. The UVA-potentiated DNA damage by the Fenton reaction system was then used to examine the protective effect of para-aminobenzoate (PABA), a UVB-absorbing sunscreen that protects against photocarcinogenesis in hairless mice. The results show that PABA and mannitol dose-dependently inhibited the damage with concentrations required for 50% inhibition at 0.1 mM and 3 mM, respectively. The protection by PABA was attributed to its radical-scavenging ability because PABA does not absorb light in the UVA region. These findings may be relevant to the biological damage by UVA and suggest that PABA is useful in protection against photocarcinogenesis by wide-range UV radiation.

Mutational specificity of 2-amino-3-methylimidazo-[4,5-f]quinoline in the hprt locus of CHO-K1 cells.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a food carcinogen formed in cooked meats, can induce gene mutation at the hprt locus of CHO-K1 cells in the presence of hepatic S9 mix. To elucidate the molecular nature of IQ-induced mutation, we characterized the entire coding region of the hypoxanthine phosphoribosyl-transferase gene of 23 independent mutants derived from IQ-treated CHO cells by direct sequencing of polymerase chain reaction-amplified cDNA. Ten of the 23 IQ-induced mutants examined contained single base substitutions; one mutant had three single-base substitutions. Among the base substitutions, G.C-->C.G (six of 13) and A.T-->C.G (three of 13) transversions predominated. Most of the base-substitution mutations occurred preferentially at a middle G and had a dA in their 3' ends. Of the 13 other mutations (56.5%), 12 missing one or more complete exons were splice-site mutations, and one mutant had a partial deletion of an exon. A high frequency of complete exon deletion (11 of 12) in exons 2-5 was observed. Interestingly, 75% of the mutants (nine of 12) with splice-site mutations were induced by IQ only at higher concentrations (300-500 microM). This was probably due to the occurrence of GC base-substitution mutations that affected hprt mRNA splicing, especially at the intron-exon boundaries.