Anthracene-9,10-diones as potential anticancer agents: bacterial mutation studies of amido-substituted derivatives reveal an unexpected lack of mutagenicity

Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254. Six of the compounds were also tested in S. typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls. Mitoxantrone was mutagenic to S. typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant. By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined. Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.     

The genotoxicity and carcinogenic potential of gastrofenzin

Genotoxicity of the Bulgarian drug gastrophensin was studied by using a battery of two genotoxicity assays "in vitro" - Salmonella/mutation assay and "in vivo" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" was tested in five mutant, histidine auxotrophic strains - TA 1535, TA 1537, TA 1538, TA 98 and TA 100 without and in the presence of metabolic activation (+/- S9) at concentration of 0.4, 2 and 10 mg center dot ml-1. Gastrophensin did not induce mutagenic response in the Salmonella/mutation assay in a range of tested concentrations in both series of assays (+/- S9). Gastrophensin did not induce micronuclei in bone marrow cells of male C57Bl6 mice at 24, 48 and 72 hours after single oral treatment with 236 mg center dot kg-1 (80% DL50 oral, mice) and 118 mg center dot kg-1 (40% DL50 oral, mice). Based on the present data a conclusion of the lack of mutagenicity and of carcinogenic potency of gastrophensin was made.     

Homodimers of the 60 kDa phosphatidylinositol-anchored transforming growth factor-beta 2 binding proteins in FBHEC and MG-63 cells

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems

Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules leading to electrophilic cations which may form adducts with DNA and other important cellular structures. Since endogenous sulfotransferases do not appear to be expressed in indicator cells of standard mutagenicity tests, rat and human sulfotransferases have been stably expressed in his- Salmonella typhimurium strain TA1538 and Chinese hamster V79 cells. Using these recombinant indicator cells, sulfotransferase-dependent genotoxic activities were detected with N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene (in the presence of co-expressed rat cytochrome P450 1A2), hycanthone, 1'-hydroxysafrole, alpha-hydroxytamoxifen and various benzylic alcohols derived from polycyclic aromatic hydrocarbons. In several cases, it was critical that the reactive sulfuric acid conjugates were formed directly within the indicator cells, owing to the inefficient penetration of cell membranes. In other cases, spontaneous benzylic substitution reactions with medium components, such as halogenide ions or amino acids, led to secondary, membrane-penetrating reactive species. Different sulfotransferases, including related forms from rat and human, substantially differed in their substrate specificity towards the investigated promutagens. It is known that some sulfotransferases are expressed with high tissue and cell type specificities. This site-dependent expression together with the limitations in the distribution of reactive sulfuric acid conjugates may explain organotropic effects of compounds activated by this metabolic pathway.     

Lack of comutagenicity by norharman and other compounds on revertants induced by photolabeling with 2-azido-9-fluorenone oxime

Mutations were induced by photolabeling 2-azido-9-fluorenone oxime in Salmonella typhimurium tester strain TA1538. Since the critical adducts were formed directly by the photogenerated nitrene, metabolic activation was bypassed. The bacteria themselves possessed the deep rough cell wall and uvrB- mutations, thereby minimizing comutagenic effects resulting from transport or DNA repair. This technique thus permitted detection of comutagenicity resulting from alteration in the binding of the mutagen to its critical receptor. No compound tested increased mutagenicity. Cholic acid had no effect at low dose, but inhibited mutagenicity at 10(-4) M, while trans-retinol no effect at any concentration. Harman, norharman and ethidium reduced the number of mutants only slightly at concentrations from 10(-6) to 10(-5) M.     

Comparison of T-cell subpopulations in cats naturally infected with feline leukaemia virus or feline immunodeficiency virus

The antimutagenicity of the Citrus flavonoids naringin, hesperidin, nobiletin, and tangeretin against the mutagens benzo[a]pyrene, 2-aminofluorene, quercetin, and nitroquinoline N-oxide was investigated in the Salmonella/microsome assay. Naringin and hesperidin showed a weak antimutagenic activity against benzo[a]pyrene. Tangeretin was antimutagenic against all indirectly-acting mutagens tested, but in general a large molar excess was necessary. Liquid preincubation increased the antimutagenicity of tangeretin against 2-aminofluorene. Nobiletin acted as an antimutagen against benzo[a]pyrene, but it enhanced the mutagenicity of 2-aminofluorene. However, in a liquid preincubation assay nobiletin also exhibited antimutagenicity against 2-aminofluorene. Both tangeretin and nobiletin inhibited the mutagenicity of quercetin. Quercetin itself acted as an antimutagen against 2-aminofluorene in a Salmonella strain (TA1538) where its mutagenicity was not expressed. Quercetin should not merely be regarded as a genotoxic risk factor in the human diet, since its mutagenicity may be inhibited by accompanying compounds including other flavonoids, and since quercetin itself also exhibits an antimutagenic action. Because of the antimutagenic properties the Citrus flavonoids tested, especially tangeretin and nobiletin, might play a role in the chemoprevention of cancer.     

Genotoxicity of Farbasol and its component: 4-ethyltoluene

A combination of assays for gene mutations in Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains with and without rat liver activation, and for micronucleus and sister chromatid exchange (SCE) in bone marrow cells of Imp:Balb/c mice was used to provide data on the mutagenic and genotoxic properties of the mixture of aromatic solvents, known under the trade name of Farbasol. In addition, 4-ethyltoluene (the main ethylmethylbenzenic component of Farbasol) was also tested for muta- and genotoxicity. The results revealed that neither Farbasol nor 4-ethyltoluene induced an increased reverse mutation in bacterial cells or the formation of micronucleated polychromatic erythrocytes in bone marrow. However, those compounds were found to be active as sister chromatid exchange (SCE) agents.     

Inflammatory pulmonary pseudotumor in an adolescent

Among p-phenylenediamine, benzidine and the analogues we previously tested, only the nitro-group containing 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenediamine, 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine caused base-pair reversion in the histidine locus of Salmonella typhimurium TA100. In order to determine the types of mutations involved, such as transversion or transition, these four nitro-group containing compounds were tested with S. typhimurium strains TA100, TA104, TA4001 and TA4006. Dose-mutagenicity relationships occurred with TA100 and TA104. However, the majority of revertants from TA100 and TA104 were insensitive to inhibition by histidine analogue, DL-1,2,4-triazole-3-alanine. These results suggested that the occurrence of histidine revertants was predominantly induced by base-pair (point) mutations and not by suppressor gene mutations. The CG-TA transition and CG-AT transversion are the major types of mutation induced by all these compounds in TA100. The TA-AT transversion also contributed to the mutagenicity of 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine in TA104. These nitro-group containing compounds showed no mutagenicity in TA4001, but induced CG-GC transversion in TA4006.     

Mutagenicity of azo dyes used in foods, drugs and cosmetics before and after reduction by Clostridium species from the human intestinal tract

Various azo dyes currently approved by the US FDA for use in foods, drugs and cosmetics are reduced by anaerobic bacteria from the human intestinal tract. These bacteria with azoreductase activities include several Clostridium species. Seven of these azo dyes and their reduction products following incubation with a Clostridium sp. were evaluated for mutagenicity in Salmonella typhimurium strains TA98 and TA100. No mutagenicity was induced in either TA98 or TA100 by any of the seven azo dyes or the reduced metabolites when tested at concentrations as high as 200 microg/plate, with or without exogenous metabolic activation by rat liver fraction S-9.     

Metabolic activation of 2-aminofluorene by isolated rat liver cells through different pathways leading to hepatocellular DNA-repair and bacterial mutagenesis

The aromatic amine 2-aminofluorene (2-AF) is metabolised by isolated rat liver cells to reactive species, thereby causing mutagenic effects in Salmonella typhimurium TA 1538 and evoking DNA-excision repair within the liver cells. The pathway leading to the production of metabolites mutagenic in Salmonella is likely to proceed via direct N-hydroxylation of 2-AF to N-hydroxy-2-aminofluorene (N-OH-2-AF). On the other hand, the formation of intermediates giving rise to hepatocellular DNA-repair is shown to depend upon N-acetylation of 2-AF to 2-acetylaminofluorene(2-AAF), whereas a subsequent conjugation reaction, most likely to be sulfate ester formation, is also essentially involved.     

Recent update on the PPAR alpha-null mouse

The most important commercially available nitro- and aminobenzenes and the explosive trinitrobenzene were tested for mutagenicity in the Salmonella typhymurium TA 98 and TA 100 both in the absence and presence of S 9. Ten of the 14 compounds tested (71%) were mutagenic. All the substances showed positive results in TA 98 and 4 substances were also mutagenic in TA 100. The three diaminobenzenes and 4-nitroaniline were mutagenic only with metabolic activation. All other compounds did not require the addition of S 9. Only nitrobenzene, 1,2-dinitrobenzene, aniline and 2-nitroaniline were negative in both strains. In summary, all substances that are derived from nitrobenzene or aniline by addition of a nitro group in the meta- or para-position were mutagenic, whereas nitrobenzene and aniline themselves and their ortho-derivates were nonmutagenic. The possible relationships between the position of the substituents and the mutagenicity are discussed.     

Mutagenicity of mixtures of halogenated aliphatic hydrocarbons and polycyclic aromatic hydrocarbons in the Ames test with TA98 and TA100

Within the framework of the assessment of the genotoxic potential of environment samples the Salmonella-microsome-test (Ames-test) is often used as a screening-test. It is one of the most applied biotest systems and possesses a large scientific acceptance. Because most environment samples are mixtures of various substances, possible effects resulting from the combination should be taken into account with regard to the mutagenic potential. In this context we investigated eight polycyclic aromatic hydrocarbons each combined with six halogenated aliphatic hydrocarbons as to their mutagenicity in the Salmonella-microsome-test with TA98 and TA100. For an exogenous metabolizing system, Arochlor 1254 induced rat liver S9-mix was used. Benz-a-pyrene in combination with bromodichloromethane (Ames neg. in TA98 and TA100 +S9) showed an increase in the number of the revertants up to 25% in TA98 and TA100 (+S9). Carbon tetrachloride (Ames neg. in TA98 and TA100 +S9) showed in TA100 (+S9) an increase in the number of the revertants of 18% at most. In the combination 3-methylcholanthrene with dichloromethane the number of revertants in TA98 (+S9) increased by 25% and in TA100 (+S9) by 18%. Hexachloroethane (weakly mutagenic in TA98 +S9) in combination showed in TA98 (+S9) a slightly increased number of revertants with benz-a-pyrene as well with 3-methylcholanthrene. All the other substances tested (chrysene, phenanthrene, anthanthrene, dibenz-a, i-pyrene, triphenylene, fluoranthene) in combination with either tetrachloroethylene or trichloroethene did not cause an increase in mutagenicity.     

Newer oral contraceptives and the risk of venous thromboembolism

Over the past 5 years, a large collaborative study of chemically-induced mutation has been performed using the four bacterial strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 in order to compare the specific spectrum of response to chemicals and to evaluate the usefulness (sensitivity) of each strain. Following the two collaborative studies to test the chemicals in category 1, chemicals previously judged as positive only in E. coli WP2 strains and derivatives of these chemicals, and category 2, oxidative agents or crosslinking agents, 22 compounds of category 3 consisting of 10 nonmutagenic carcinogens and another 12 chemicals were selected in this study. Twenty participating laboratories tested each compound in the same method as previous reports. In the group of nonmutagenic carcinogens, no chemical induced revertant colonies of any strain tested. In the group of other chemicals, response to the chemicals was similar in TA102 and WP2 uvrA/pKM101. Overall, in the three collaborative studies, a total of 79 compounds were tested. No difference in qualitative response to the four strains was observed for 71% (56/79) of the test chemicals. The combination of strains providing the greatest number of positive responses was WP2 uvrA/pKM101 with TA102; 84% (66/79) of the test chemicals elicited the same qualitative response in these two strains. Therefore, it is suggested that WP2 uvrA/pKM101 and TA102 can be included as a part of the standard tester strains for detection of mutagenic activity of chemicals.     

工业纯钛TA2冷轧板再结晶过程的研究

利用Gleeble-3500热模拟试验机对工业纯钛TA2冷轧板进行退火实验,结合硬度法与金相法测定了TA2的再结晶温度,根据Arrhenius公式计算了TA2的再结晶激活能,并对冷轧退火板进行力学性能测试。结果表明保温时间为9 min时,TA2的再结晶温度在520～600℃之间,恒温700℃时,再结晶时间为1.84 min;再结晶激活能Q为5.6578×104 kJ.mol-1;当再结晶退火温度在680～700℃,保温时间在30min左右,钛板可以保持良好的力学性能。     

Primary biliary cirrhosis: an electronic diagnostic tool trial based on symptoms, (past) history and signs only, using the European database euricterus. The Euricterus PMG

BACKGROUND/AIMS: Primary Biliary Cirrhosis (PBC) is a relatively rare chronic progressive disease in which a working diagnosis of PBC easily leads to a final diagnosis by testing for anti-mitochondrial antibodies. Liver transplantation is the only effective treatment. The aim of this study was to test an electronic diagnostic tool (tool) for it's ability to include PBC in the working differential diagnosis. METHODOLOGY: In the European Union Euricterus project a large number of (sub)icteric patients in 17 discrete disease categories, PBC being one of them, were gathered prospectively. A tool was developed-using Bayes (B) and Trial Algorithm (TA) pattern-recognition and based on items related to the history, symptoms and signs of all Euricterus patients. We have tested the diagnostic tool on 143 PBC Euricterus patients. RESULTS: PBC was mentioned by the tool in 86% (B) and 91% (TA) of the 143 patients. These figures were higher for patients under 60 and (TA only) females. Females under 60 (n = 89) scored 92% B and 96% TA. A sole diagnosis of PBC was made in 31% (B) and 66% (TA). In the other patients with a PBC probability, 7 other (first) diagnoses were presented by the tool of which non-alcoholic active liver disease and pancreatic or biliary carcinoma were the leaders. These 7 diseases appeared evenly distributed along the percentual probabilities of PBC given by the tool (B) and also along Pugh and Mayo scores (B and TA). PBC was mentioned by the tool in all patients with a Pugh score 10 or higher (advanced disease, class C). In the patients in whom the tool did not mention PBC, the primary diagnoses came from 9 other disease categories. CONCLUSION: This electronic tool has been able to identify PBC as one of the differential diagnostic modalities in the large majority of a present population of PBC patients.     

Increasing the moment arm of the tibialis anterior induces structural and functional adaptation: implications for tendon transfer

Previous studies on the functional effects of tendon transfer have not examined possible muscle adaptation following transfer. The purpose of the present study was to test the hypothesis that muscle adapts to increased moment arm and excursion such that joint torque is maintained near normal levels. The moment arm and excursion of the tibialis anterior (TA) were increased by releasing the TA from its retinacular restraint at the ankle joint in growing (4-week-old) rabbits. Twelve weeks post-release, in vivo TA force during hopping was smaller in released compared with control rabbits, compensating for the increased moment arm, and thus TA torque at the ankle joint was not significantly different between groups. Physiological cross-sectional area was smaller, and the number of sarcomeres in series was larger, in the released TA compared with the control TA. These adaptations may result from chronically decreased in vivo TA force production, and chronically increased TA excursion, respectively. In addition, these adaptations were consistent with the smaller in vivo force for the released TA. Comparisons between control and sham-operated rabbits showed no significant differences for in vivo TA force, torque, or muscle architecture. Thus, muscle appears capable of adapting to increased moment arm and excursion such that joint torque is maintained near normal levels. These findings have important implications for tendon transfer procedures that increase the moment arm and/or excursion of the released muscle.     

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/beta NP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK+/- cells when tested to the limits of its solubility in the culture medium (+/- S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.     

Polymyalgia rheumatica and temporal arteritis: a retrospective study of 111 patients

There is no unanimity as to whether polymyalgia rheumatica (PMR) and temporal arteritis (TA) are two distinct diseases or different features of one disease. The objective of this study was to assess the value of histological findings of temporal artery biopsy and the efficacy and complications of drug therapy as well as the frequency of malignancies. It was carried out as a retrospective follow-up study. One hundred eleven patients (89 PMR, 14 TA and 8 PMR+TA) were studied. In 56 patients with PMR a temporal artery biopsy was performed; in none of these biopsies was active arteritis found. Of the 19 patients with TA or PMR+TA, where a temporal artery biopsy was performed, arteritis was found in 15 patients. Reactivation occurred in 27 patients: 4 patients using NSAIDs and 23 patients using corticosteroids. Side effect of the medication included vertebral compression in 10 patients, most of whom were using corticosteroids. Malignancies were diagnosed in 12 of the 111 patients. Most malignancies were diagnosed long before or after the diagnosis of PMR. In case of a PMR diagnosed by the clinician a biopsy of the temporal artery has no value, while the yield of this diagnostic procedure is high in TA. Reactivation was seen quite often and warrants a prolonged period of medical treatment.     

Test anxiety and the hierarchical model of approach and avoidance achievement motivation.

This research was designed to incorporate the test anxiety (TA) construct into the hierarchical model of approach and avoidance achievement motivation. Hypotheses regarding state and trait TA were tested in 2 studies, and the results provided strong support for the predictions. State TA (specifically, worry) was documented as a mediator of the negative relationship between performance-avoidance goals and exam performance. The positive relationship between performance-approach goals and exam performance was shown to be independent of TA processes. A series of analyses documented the conceptual and functional convergence of trait TA and fear of failure (FOF), and further validation of the proposed integration was obtained by testing trait TA/FOF and state TA together in the same model. Mastery goals were positively and performance-avoidance goals negatively related to long-term retention. (PsycINFO Database Record (c) 2010 APA, all rights reserved)