Genetic effects of the decay in Saccharomyces cerevisiae yeast cells of the radionuclide products of nuclear fuel fission. II. Lethal mutagenic effects and the nature of mutations induced by an equilibrium mixture of 90Sr-90Y and 89Sr

The lethal and mutagenic effects and the nature of mutations induced by 90Sr-90Y and 89Sr in cells of the yeast Saccharomyces cerevisiae were studied. The lethal efficiency was determined for 89Sr as (7,6 +/- 1,05) X 10(-5) decay-1, for 90Sr-90Y-(3,3 +/- 1,6) X 10(-4) decay-1. The mutagenic efficiency for ade1 and ade2 genes was determined for 89Sr as (8,3 +/- 2,5) X 10(-9) decay-1, for 90Sr-90Y-(2,9 +/- 1,5) X 10(-8) decay-1. For ade2 locus, the spectrum of mutations induced by 89Sr was a follows: one deletion, 17% of frameshifts and 83% of base pair substitutions--51% of transversions, 22% of GC-AT transitions and 10% of AT-GC transitions. The data of the present work suggest that 90Sr-90Y and 89Sr are very efficient physical mutagens. The relative mutagenic efficiency (RME) was estimated for radionuclides studied.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Mutagenic effects of gamma-rays and incorporated 8-3H-purines on extracellular lambda phage: influence of mutY and mutM host mutations

The lethal and mutagenic effects on phage lambdacI857 of 60Co gamma-rays and of decay of 3H incorporated into phage DNA both as 8-3H-deoxyadenosine and 8-3H-deoxyguanosine (using 8-3H-adenine as a labelled DNA precursor) were studied on four isogenic Escherichia coli strains: AB1157 M(+)Y(+) (wild type, mutM(+) mutY(+)), AB1157 M(-)Y(+) (mutM::kan mutY(+) mutant deficient in the formamidopyrimidine-DNA glycosylase MutM), AB1157 M(+)Y(-) (mutM(+) mutY mutant deficient in the A:G mismatch DNA glycosylase MutY), and AB1157 M(-)Y(-) (mutM::kan mutY double mutant deficient in both DNA glycosylases). The main products of transmutation component of 3H decay in position 8 of purine residues are 8-oxo-7, 8-dihydroadenine (8-oxoA) and 8-oxo-7,8-dihydroguanine (8-oxoG), the latter being responsible for the most part of the mutagenic effect. The lethal effects of both gamma-rays and tritium decay virtually did not depend on the repair phenotypes of the host strains used. Therefore, the MutM and MutY glycosylases are not involved in the repair of lethal DNA damages induced by ionizing radiation or by the transmutation component of 3H decay in purine residues of phage DNA. The efficiencies of mutagenic action of 3H-purines E(m) (frequencies of c-mutations per one 3H decay in phage genome) were 2.4-, 3.8- and 55-fold higher in the M(-)Y(+), M(+)Y(-) and M(-)Y(-) mutants, respectively, in comparison to the wild-type host. The mutagenic efficiencies E(m) for gamma-rays were nearly identical in the M(+)Y(+) and M(-)Y(+) hosts, but were increased 1.8- and 8.3-fold, respectively, in the M(+)Y(-) and M(-)Y(-) mutants. These data suggest that: (1) the MutY and MutM DNA glycosylases are important for prevention of mutations caused not only by spontaneous oxidation of guanine residues, but also by ionizing radiation or by decay of 3H incorporated into purine bases of DNA; (2) the MutY and MutM enzymes functionally cooperate in elimination of mutagenic damages induced by these agents.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Lethal and mutagenic action of incorporated 5-3H-cytosine on extracellular phage lambda

A study was made of the lethal and mutagenic effects on extracellular phage gamma of 5-3H-cytosine incorporated into DNA. The efficiencies of inactivation by incorporated 3H were equal for 5-3H-cytosine and [3H-methyl]-thymidine, but the yield of c-mutations for the former was 14 times higher. The lethal and mutagenic effects of incorporated 5-3H-cytosine did not depend on ung mutation of host cells which caused a deficiency in uracil-DNA-glycosylase. The mutagenic effect was not enhanced when SOS-repair system was induced by UV-radiation. The mutagenic effect of 5-3H-cytosine was associated with the modified mispairing bases but not with uracil residues.     

Efficient Recovery of Enu-Induced Mutations from the Zebrafish Germline

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F(1) progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 X 1 hr in 3 mM ENU and 6 X 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F(2) screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.     

Molecular nature of the direct mutations induced by 4-nitro-quinoline-N-oxide and 3-methyl-4-nitroquinoline-N-oxide in the ADE2 gene of Saccharomyces cerevisiae

The lethal and mutagenic effects and the nature of forward mutations in ADE2 gene induced by highly carcinogenic agent 4-nitroquinoline-N-oxide (4NQO) and its noncarcinogenic analogue 3-methyl-4-nitroquinoline-N-oxide (3M4NQO) have been examined in Saccharomyces cerevisiae. It is shown that 3M4NQO is more toxic than 4NQO. Both are very efficient mutagens: the mutagenic efficiency for ADE1 and ADE2 genes was 7.9 X 10(-5) for 4NQO and 10.5 X 10(-5) for 3M4NQO. The base pair substitutions are the main type of induced mutations in ADE2 gene (95 and 89% for 4NQO and 3M4NQO, respectively); among these 40% transversions for 4NQO and 63% for 3M4NQO, GC----AT transitions-32 and 31% for 4NQO and 3M4NQO, respectively, AT----GC transitions-23 and 22% for 4NQO and 3M4NQO, respectively. The results obtained indicate that 4NQO and 3M4NQO induce the same spectrum of mutations in ADE2 gene and that both mutagens are nonspecific in yeast cells.     

A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar

A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 +/- 3.2 X 10(-6) and cloning efficiencies of 91 +/- 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the CHO/HGPRT mutation assay.     

Mutagenicity of lead chromate in Drosophila melanogaster in the presence of nitrilotriacetic acid (NTA)

By using the sex-linked recessive lethal mutation test in Drosophila melanogaster (standard Basc scheme) we analysed the mutagenic effects of treatments by feeding with nitrilotriacetic acid (NTA: 5 X 10(-2) M), with the insoluble Cr(VI) compound lead chromate, PbCrO4 (supernatant of 4.6 X 10(-4)-M suspension in which the actual concentration was 0.06 gamma/ml as Cr(VI)) and with both compounds preincubated at 3 relative ratios (NTA: 5 X 10(-2) M; PbCrO4: 4.6 X 10(-4), 4.6 X 10(-5) and 9.2 X 10(-6) M, respectively). The estimation of mutation frequencies was done at different developmental stages of the germ cells, namely spermatozoa, spermatids and spermatocytes. Ethyl methanesulphonate (EMS: 5 X 10(-3) M) was used as the reference positive control, with clearly mutagenic results. Treatments with NTA or with PbCrO4 alone did not induce any significant increase of the mutation frequency. PbCrO4 at the 3 concentrations tested was completely soluble in the 5 X 10(-2)-M NTA solution, and the mixture of NTA and PbCrO4 induced significant increases of the frequency of sex-linked lethal mutations, with a significant dose-effect relationship with respect to PbCrO4, apparently as a result of the interaction of the compounds and subsequent release of the genotoxic heavy-metal Cr(VI) ions. This result indicates an important synergistic action of NTA with PbCrO4 under the conditions described.     

Identification and Characterisation of 5-Hydroxytryptamine3 Recognition Sites in Human Brain Tissue

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.     

The lethal and mutagenic action of 3H incorporated into the 2' position of the deoxyribose in the DNA of the extracellular phage lambda]

The lethal and mutagenic effects of 3H decay in 2' position of deoxyribose residues in DNA of extracellular lambda phage were studied, [2'-3H]-deoxyadenosine (3H-dA) or [2'-3H]-thymidine (3H-dT) being used as labelled DNA precursors. As estimated by the efficiency of the lethal and mutagenic actions of 3H decay in position 2' was significantly lower than that of the decay in the incorporated 3H-pyrimidines. The genetic effects of 3H decay in 2' position may be attributed to the radiation effect of beta-particles on DNA. In UV-irradiated E. coli cells, with the induced SOS repair, the mutagenic effect of 3H-dA in phage lambda is significantly higher than that of 3H-dT. This is perhaps related to the formation in DNA of AP-sites, resulting from 3H-decay in 2' position, and to the predominant incorporation of adenosine residues opposite to AP-sites during SOS repair.     

Role of high-density lipoprotein in transport of circulating bilirubin in rats

The analbuminemic rat strain established by Nagase et al. (Nagase, S., Shimamune, K., and Shumiya, S. (1979) Science 205, 590-591) exhibits hereditary deficiency in albumin biosynthesis. Serum bilirubin concentration is rather lower in homozygous (aa) rats (0.009 +/- 0.002 mg/dl) as compared with heterozygous (Aa) rats (0.047 +/- 0.009 mg/dl) or wild-type Sprague-Dawley (AA) rats (0.034 +/- 0.006 mg/dl) as evidenced by high pressure liquid chromatography analysis of bilirubin. After intravenous administration of various amounts of [heme-3H]hemoglobin in rats, [3H]bilirubin derived from [3H]heme of hemoglobin in vivo is more efficiently excreted into bile in aa rats than in Aa or AA rats. [3H]Bilirubin is exclusively bound with high-density lipoprotein (HDL) in aa rats, and a significant amount of [3H]bilirubin is shown to bind with HDL in Aa or AA rats in vivo. Scatchard plots revealed that [3H]bilirubin is bound with HDL in three binding modes depending on the molar ratio of [3H]bilirubin to HDL: Kd = 0.8 X 10(-7) M (molar ratio, 0.02-0.06), Kd = 1.6 X 10(-6) M (molar ratio, 0.06-0.41), and Kd = 1.2 X 10(-4) M (molar ratio, 0.79-9.02). Even under extreme conditions of excess hemoglobin administration, the molar ratio remains under 0.041; and thus, expected the Kd value would remain around 0.8 X 10(-7) M. Binding of [3H]bilirubin to rat serum albumin revealed two distinct binding modes depending on the molar ratio of [3H]bilirubin to rat serum albumin: Kd = 3.6 X 10(-7) M (molar ratio, 0.03-0.21), and Kd = 5.0 X 10(-6) M (molar ratio, 0.21-2.46). Under physiological conditions in Aa or AA rats, the former mode would be more reliable than the latter. Thus, HDL could bind with approximately 4.5 times higher affinity than rat serum albumin in Aa or AA rats under physiological conditions in vivo.     

Studies of angiotensin I converting enzyme: effects of kinins, bacitracin, gamma-aminobutyric and epsilon-aminocaproic acids, and related compounds on substrate binding and catalysis in vitro

Bradykinin and 22 of its analogs were evaluated for their abilities to inhibit the hydrolysis of [3H]hippurylglycylglycine by purified porcine kidney angiotensin I converting enzyme. The mean inhibitory concentration (IC50) for bradykinin was 1.2 +/- 0.2 X 10(-6) M. Except for Ile-Ser-bradykinin and [Sar4]-bradykinin, none of the kinin analogs were more potent in this regard than bradykinin. Bacitracin, gamma-aminobutyric acid, epsilon-aminocaproic acid, and structurally related compounds were also tested. The IC50 value for bacitracin was 1.9 +/- 0.4 X 10(-4) M, gamma-aminobutyric acid, 83.4 +/- 7.2 mM, and for epsilon-aminocaproic acid, 7.0 +/- 1.4 mM. Compounds were also evaluated for their abilities to prevent 125I-labelled [Tyr1]-kallidin binding to angiotensin I converting enzyme inhibited by EDTA. The IC50 values for bradykinin, bacitracin, gamma-aminobutyric acid, and epsilon-aminocaproic acid were 1.6 +/- 0.3 X 10(-8) M, 2.6 +/- 0.9 X 10(-6) M, greater than 291 mM, and 13.2 +/- 3.9 mM, respectively.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

Lethal and mutagenic effects photoinduced in haploid yeast (Saccharomyces cerevisiae) by two new monofunctional pyridopsoralens compared to 3-carbethoxypsoralen and 8-methoxypsoralen

The photobiological effects of two monofunctional pyridopsoralens (PPs), pyrido[3,4-c]psoralen and pyrido[3,4-c]-7-methylpsoralen were studied and compared to those of 3-carbethoxypsoralen (3-CPs) and 8-methoxypsoralen (8-MOP) in a haploid wild-type strain of yeast (Saccharomyces cerevisiae). The capacity of PPs to photoinduce lethal effects in the presence of 365-nm radiation was not only higher than that of the monofunctional compound 3-CPs, but also higher than that of the bifunctional compound 8-MOP. This activity was apparently independent of oxygen, and it was found that it was probably due to the induction of monoadducts in DNA. A high effectiveness of PPs on the induction of cytoplasmic 'petite' mutations was observed suggesting a high photoaffinity towards mitochondrial DNA. In contrast to 8-MOP, the strong cell killing activity of PPs was not accompanied by a strong inducing effect on nuclear mutations (HIS+ reversions or canR forward mutations). For these endpoints, PPs were less effective per unit dose of 365-nm radiation and also less efficient per viable cell than 8-MOP. From this, it appears that the lesions photoinduced by the former compounds show a more lethal than (nuclear) mutagenic potential. Furthermore, the fact that PPs were even less mutagenic (nuclear) per viable cell than the monofunctional compound 3-CPs suggests that the activity of these agents may differ in frequency and nature of lesions induced. The photobiological activity of PPs in haploid yeast appears to be in line with the recent proposition for their use in photochemotherapy.     

Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxic effects in diploid yeast

The induction of genetic effects was studied in a diploid strain of Saccharomyces cerevisiae (D7) after treatments with the monofunctional furocoumarins 7-methylpyrido[3,4-c]psoralen (MePyPs), pyrido[3,4-c]psoralen (PyPs) and 3-carbethoxypsoralen (3-CPs) and the bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of 365-nm radiation. The DNA photobinding of radioactively labelled MePyPs, 3-CPs, 5-MOP and 8-MOP was determined in parallel. The DNA-photobinding capacity was highest for MePyPs followed in decreasing order by 5-MOP, 3-CPs and 8-MOP. At a concentration of 5 microM and 4.2 kJ/m2 of 365-nm radiation approximately 160, 66, 60 and 16 adducts per 10(6) base pairs were formed by MePyPs, 5-MOP, 3-CPs and 8-MOP, respectively. The activity of MePyPs and PyPs for the induction of lethal effects lay in the same range as that of 5-MOP whereas 8-MOP was 3 times less active and 3-CPs showed very little activity. For the induction of mitotic gene conversion and genetically altered colonies including mitotic crossing-over the order of activity was about the same as that observed for the induction of lethal effects: MePyPs greater than 5-MOP greater than PyPs greater than 8-MOP much greater than 3-CPs. Nuclear reversions were induced most effectively by 5-MOP, 8-MOP being about 3 times less effective. Up to 4 and 6 kJ/m2 of 365-nm radiation, MePyPs and PyPs, respectively, were less mutagenic than 8-MOP but became more mutagenic at higher doses. At equal survival, the pyridopsoralens were, however, clearly less mutagenic than the bifunctional furocoumarins 8-MOP and 5-MOP. By plotting the genetic data versus the number of lesions induced in DNA, it was shown that the monoadducts induced by the monofunctional furocoumarins MePyPs and 3-CPs exert a relatively low potential for the induction of lethal and nuclear genetic events as compared to photoadditions induced by the bifunctional furocoumarins 8-MOP and 5-MOP. However, at a very high density, the monoadducts induced by MePyPs became as lethal and as mutagenic as the mixture of mono- and biadducts induced by 8-MOP and 5-MOP probably due to overloading of cellular repair capacities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets.

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82 X 10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87 X 10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.     

Molecular dosimetry of the mutagen ethyl methanesulfonate in Drosophila melanogaster spermatozoa: linear relation of DNA alkylation per sperm cell (dose) to sex-linked recessive lethals.

The dosage-response curve for EMS was determined with dose measured as ethylations of DNA per sperm cell, and response measured as the relative frequency of sex-linked recessive lethals induced in sperm cells of Drosophila melanogaster. Dose can be converted to ethylations per nucleotide of DNA by dividing ethylations of DNA per sperm cell by 3 X 10(8) nucleotides per sperm cell. Adult males were exposed to equal amounts of either [3H]EMS for determining dose or nonlabeled EMS for determining mutational response. By feeding EMS for 24 h in a concentration of 25 mM, a high dose of 1.4 X 10(-2) ethylations per nucleotide was observed. With 1.4% of the nucleotides ethylated, 57% of the X-chromosomes were hemizygously viable; therefore, ethylation per se is not very efficient in inducing mutations. The relative frequency of mutations increased linearly with the dose from a dose of 2.1 X 10(-4) to 1.4 X 10(-2) ethylations per nucleotide. No threshold was apparent, and the statistical limits of the exponent, 1.0 +/- 0.1, excluded an exponent as high as 1.2. This linear relation suggests no change in mechanism of mutagenesis occurs from low to high dose in Drosophila. A nonlinear relation was found between exposure and dose; when exposure was increased by a factor of 250 (from 0.1 to 25 mM EMS in the feeding medium) dose was increased by a factor of only 68. By extrapolating down from our lowest dose of 2.1 X 10(-4) ethylations per nucleotide with an observed frequency of 0.55% +/- 0.08% sex-linked recessive lethals, we estimate the doubling dose for sex-linked recessive lethals to be 4 X 10(-5) ethylations per nucleotide.