Mutagenicities of the pyrolyzates of peptides and proteins.

Pyrolyzates of 10 peptides, 10 proteins and 5 naturally-occurring materials were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Significant mutagenic activity was detected with pyrolyzates of most of these materials. The pyrolyzates requred a liver microsomal fraction, as representative of mammalian metabolism, for their detection as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of a tryptophan-containing peptide. The pyrolyzate of protein obtained from tobacco leaf also showed mutagenicity. The higher the protein content in the leaf the higher the mutagenic activity of the pyrolyzate. Protein in a tobacco leaf may be the principal precursor of mutagens in tobacco-smoke condensate.     

The effect of hycanthone and maleic hydrazide on the frequency of micronuclei in the bone-marrow erythrocytes of mice.

Male Swiss mice were assigned to 6 groups of either 3 or 4 animals each. 3 groups were given hycanthone methanesulfonate intraperitoneally, at 40, 80 or 120 mg/kg, respectively; the dose was repeated after an interval of about 24 h. At the same time 2 groups received maleic hydrazide at 100 or 200 mg/kg, and the remaining group was given dimethyl sulfoxide which was used as a solvent for both drugs. 6 h after the second injection, the mice were killed and bonemarrow preparations were made. Hycanthone induced a significant increase in the frequency of micronuclei in the polychromatic erythrocytes and suppressed the P/N ratio significantly. However, there was no dose-response relationship. Maleic hydrazide, on the other hand, failed to influence the incidence of micronuclei or the ratio of poly- to normo-chromatic erythrocytes.     

Effects of methylation and ring size on mutagenicity of cyclic nitrosamines in Drosophila melanogaster

The mutagenic activity of 7 nitrosopiperazines, 2 nitropyrrolidines, and 3 nitrosomorpholines was examined in the X-linked recessive-lethal assay of Drosophila melanogaster. Mutagenicity is also reported for a series of cyclic nitrosamines that differ in structure only in the number of carbon atoms in the ring. Of the 18 compounds tested, 6 (nitrosopiperazine; 2,3,5,6-tetramethyl-dinitrosopiperazine; nitrosoproline; 2,5-dimethylnitrosopyrrolidine; nitrosothiomorpholine; and nitrosooctamethyleneimine) were nonmutagenic. As we reported earlier in investigations with the nitrosopiperidines, substitutions with methyl groups at all of the α-carbon atoms reduce or eliminate the mutagenic activity of dinitrosopiperazine and nitrosopyrrolidine.     

Genetic activity of niridazole in yeast.

Niridazole, one of several drugs presently known to be of value in the treatment of human schistosomiasis, was tested for its activity in inducing mitotic recombination in yeast. It was found that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium (YPG-medium) under growing conditions, but not when treatment is carried out in a non-nutrient suspension (phosphate buffer). The data suggest that niridazole might be converted to an active compound by yeast metabolism. The results of the experiments with niridazole in the non-nutrient medium were compared with those of AF-2 and SQ18, 506, two agents which have been shown to be genetically active in the present assay system.     

Effects of Dimethyl Sulfoxide on Tetrahymena pyriformis GL. Fine Structural Changes and Their Reversibility*

SYNOPSIS. Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.     

Differential effect of recombinant human leukocyte interferon on human leukemic and normal myeloid progenitor cells

Two separate clones of recombinant leukocyte interferon (IFLrA and IFLrD) inhibited the cloning efficiency in soft agar of the human leukemia cell lines HL-60 and KG-1. Inhibition of the growth in agar of normal human bone marrow myeloid progenitors was also observed, but this required considerably higher concentrations. IFLrA and IFLrD also inhibited the growth of HL-60 and KG-1 cells in suspension culture. This antiproliferative effect did not appear to be due to induction of maturation of these cells. Our results suggest that homogeneous preparations of interferon may be capable of exerting selective antiproliferative effects on malignant human myeloid progenitor cells in comparison to their normal counterparts.     

二甲基亚砜对离体猪皮肤组织的拉曼光谱的影响

拉曼光谱技术作为鉴定生物分子种类最有力的分析工具之一,具有快速、简单、无损、准确等优点.目前,拉曼光谱已被国内外学者广泛开展了在人体组织的应用研究,但由于生物组织具有高散射性,因此限制了拉曼光谱对其的检测深度.本文主要采用光透明剂--二甲基亚砜对组织拉曼光谱的影响进行研究,对离体猪皮组织的不同深度（100 μm,200 μm,300 μm,400 μm）拉曼光谱强度随处理前、后不同时间（0 min,10 min,20 min,30 min,60 min）的变化进行对比.结果发现,不管是经二甲基亚砜处理前还是后,都出现随着距猪皮组织表面的深度加深,其拉曼光谱强度都不断减少;同时发现各层猪皮组织随着处理后时间的加长,其特征峰的强度不断加强,信噪比逐渐提高,在处理后的60 min效果最好;而且出现了在经二甲亚砜处理前看不见的峰（1 126 cm-1和1 426 cm-1）.结果表明：5%DMSO对组织的处理能增强其拉曼光谱的强度,同时也能使拉曼光谱仪的信噪比提高,并且使组织谱图中特征峰也得到相应的增加.     

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.     

Theoretical prediction of mutagenicity and carcinogenicity of chemical substances.

The pseudopotential with respect to free electrons has been calculated for a series of chemical substances. These potentials are different for carcinogenic, mutagenic-carcinogenic, and mutagenic substances thus allowing the theoretical prediction of their biological activity. The reaction mechanism is discussed on the basis of the interaction potential.     

The relationship between mutagenicity and carcinogenicity of some nitrosamines.

The carcinogenicity and mutagenicity of 26 nitrosamines were compared to help validate the predictability of a short-term in vitro test. 80% of the compounds showed agreement between the two characteristics, while 20% did not. Of the latter group, 8% were false positives and 12% were false negatives.     

A comparison of cytotoxicity, ouabain-resistant mutation, sister-chromatid exchanges, and nascent DNA synthesis in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Chinese hamster V79 cells were treated with either (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide I) or (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide II) and the nascent DNA was labeled with [Me-3H]thymidine. The cells were harvested for determination of cytotoxicity, sister-chromatid exchanges (SCE), ouabain-resistant (Or) mutations and the size of newly synthesized daughter-strand DNA. Both isomers caused dose-dependent decreases in survival of cells and in the size of nascent DNA. Increases in the frequencies of SCE and of Or mutation were found in cells treated with either isomer. However, B[a]P-diol epoxide I caused 10--20-fold more Or mutations and 50-100% more SCE than did B[a]P-diol epoxide II at equal molar dose levels. In contrast to the marked difference in the frequencies of both SCE and Or mutations caused by both compounds, the isomers induced similar reductions in the size of the nascent DNA at equal dose levels. In comparing the molecular and biological effects of the two isomers the reduction in the size of nascent DNA was more closely related to cytotoxicity than to the induction of SCE or Or mutations.     

The relationship between carcinogenicity and mutagenicity of some polynuclear hydrocarbons.

25 polynuclear hydrocarbons were tested for mutagenicity using the Ames Assay and the results were compared with existing carcinogenicity data. The assessment of the predictive value of this particular short-term test showed a 58% positive and a 41% negative correlation.     

Mechanism of corticotropin action in rat adrenal cells I. The effects of inhibitors of protein synthesis and of microfilament formation on corticosterone synthesis

The contribution of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steriodogenesis in rat adrenal cell suspensions has been assessed by use of a series of inhibitors to each function. Five inhibitors of protein synthesis (cycloheximide, puromycin, blastocidin S, anisomycin, and trichodermin) each exhibited time-dependent inhibition of corticotropin-stimulated steroidogenesis. For the first 30 min, steroidegenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidegenesis but not on protein synthesis. The reversal effects was not observed at high levels of inhibitors. One inhibitor of microfilament fromation (cytochalasin B) and four inhinitors of microtubule formation (colchicine, podophyllotoxin, vinblastine sulfate and griseofulvin) inhibited steroidogenesis without inhibiting protein synthesis and without any reversal effect with prolonged incubation. The actions of all ten inhinitors were shown to be fully reversible. Cell superfusion of adrenal cells showed that the decay of steroidogenesis upon addition of all the protein synthesis inhibitors was similar to decay upon removal of corticotropin from the medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4–6}\text{min}$). Recoveries from inhibition upon removal of the inhibitors were similar to each other and comparable to initial corticotropin stimulation of the cells (lag of 3–5 min, $\text{f}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7–9}\text{min}$). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhbition of cytochrome P-450_sec by an aminoglutethimide was complete within 1 min and was rapidly reversed.Injection of each inhibitors (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450_sec in adrenal mitochondria isolated from these rats was also decreased by all inhbitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450_sec (r = 0.94).It is concluded that protein synthesis and steroidogenesis must be intimately coupled propbably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450_sec. An involvement of microtubules and microfilaments in this process is clearly indicated.     

Think Twice: Understanding the High Potency of Bis(phenyl)methane Inhibitors of Thrombin

Successful design of potent and selective protein inhibitors, in terms of structure-based drug design, strongly relies on the correct understanding of the molecular features determining the ligand binding to the target protein. We present a case study of serine protease inhibitors with a bis(phenyl)methane moiety binding into the S3 pocket. These inhibitors bind with remarkable potency to the active site of thrombin, the blood coagulation factor IIa. A combination of X-ray crystallography and isothermal titration calorimetry provides conclusive insights into the driving forces responsible for the surprisingly high potency of these inhibitors. Analysis of six well-resolved crystal structures (resolution 1.58-2.25 Å) along with the thermodynamic data allows an explanation of the tight binding of the bis(phenyl)methane inhibitors. Interestingly, the two phenyl rings contribute to binding affinity for very different reasons — a fact that can only be elucidated by a structure-based approach. The first phenyl moiety occupies the hydrophobic S3 pocket, resulting in a mainly entropic advantage of binding. This observation is based on the displacement of structural water molecules from the S3 pocket that are observed in complexes with inhibitors that do not bind in the S3 pocket. The same classic hydrophobic effect cannot explain the enhanced binding affinity resulting from the attachment of the second, more solvent-exposed phenyl ring. For the bis(phenyl)methane inhibitors, an observed adaptive rotation of a glutamate residue adjacent to the S3 binding pocket attracted our attention. The rotation of this glutamate into salt-bridging distance with a lysine moiety correlates with an enhanced enthalpic contribution to binding for these highly potent thrombin binders. This explanation for the magnitude of the attractive force is confirmed by data retrieved by a Relibase search of several thrombin-inhibitor complexes deposited in the Protein Data Bank exhibiting similar molecular features.Special attention was attributed to putative changes in the protonation states of the interaction partners. For this purpose, two analogous inhibitors differing mainly in their potential to change the protonation state of a hydrogen-bond donor functionality were compared. Buffer dependencies of the binding enthalpy associated with complex formation could be traced by isothermal titration calorimetry, which revealed, along with analysis of the crystal structures (resolution 1.60 and 1.75 Å), that a virtually compensating proton interchange between enzyme, inhibitor and buffer is responsible for the observed buffer-independent thermodynamic signatures.     

Dimethyl sulfoxide-induced high enantioselectivity of subtilisin Carlsberg for hydrolysis of ethyl 2-(4-substituted phenoxy)propionates

The enantioselectivity for subtilisin-catalyzed hydrolysis of ethyl 2-(4-substituted phenoxy)propionates in an aqueous buffer solution was improved by addition of DMSO (54–56% v/v). On the basis of the conformational change of subtilisin Carlsberg observed for FT-IR and CD spectra, the high enantioselectivity for subtilisin-catalyzed hydrolysis of racemic ethyl 2-(4-ethylphenoxy)propionate could be related to a partial decrease of the tertiary structure of the enzyme protein arising from an increase of the ratio of DMSO in the reaction medium. This mechanistic model for the enantiorecognition can also be supported by the discussion based on the value of the Michaelis constant (K _m) obtained for each enantiomer of the substrate.     

Evaluation of mutagenicity and carcinogenicity using three-tier system

A three-tier approach to mutagenicity evaluation has previously been proposed [1] with three underlying general principles. (1) No generally mutagenic chemical should be released into the environment or be permitted to be used if there exists a satisfactory non-mutagenic substitute; (2) the extent and rigour of the screening procedures should be related to the extent to which man is, or is likely to be exposed to the agent; (3) a mutagenic substance may be used if the benefits are judged to be great enough to outweigh the hazards and if appropriate controls are exercised. The first tier contains short-term screening tests with sub-mammalian systems, the second tier contains short- and longer-term tests with whole mammals, and the third tier involves a risk-benefit evaluation which may entail further more specialized testing procedures and experiments on the detailed metabolism of the agent in vivo.Such a scheme may in principle be used wherever there exists a long-term hazard for which short-term predicitive tests exist. Carcinogenicity evaluation fits well into a three tier approach and evaluation for carcinogenicity and mutagenicity may be run in parallel with some degree of overlap [2]. Long term carcinogenicity trials fall into tier 2, for example, while tier 1 may include (in addition to mutagenicity) such tests as degranulation of endoplasmic reticulum [3] induction of biphenyl hydroxylase [4] and transformation of cells to malignancy in vitro.The third tier of risk-benefit evaluation involves the determination of risk. This is almost impossible to do in a quantitative manner at the present time. Nevertheless some guidance must be given to the decision-maker, however approximate and surrounded by reservations it may be. One approach is to use the radiation-equivalent dose concept using data from a system that is as closely related as possible to man [1]. Such a result may have some validity for the particular end-point and dose range used. Other end-points and dose ranges may give a different value for radiation equivalence and the weight given to it must be determined by the likely importance of the end-point in constituting a genetic hazard to man. The radiation-equivalent dose is thus a common unit of convenience for chemical mutagens which also has the advantage of being more readily understood by regulators and decision-makers without a genetic background. An example of a tentative and qualified radiation-equivalent calculation has been carried out for the fungicide captan [5]. The use of radiation-equivalent concept by Committee 17 [6] seems to imply a near constancy of radiation equivalent dose values over a wide range of species and end-points. This extensio of the radiation-equivalent concept from its use as an approximate common unit of effect to a philosophy for making extrapolations between widely different species does not seem to be justified at the present time.