Institut für Pharmakologie und Toxikologie
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Patients with end-stage renal disease (ESRD), whether on conservative, peritoneal or hemodialysis therapy, have elevated genomic damage in peripheral blood lymphocytes and an increased cancer incidence, especially of the kidney. The damage is possibly due to accumulation of uremic toxins like advanced glycation endproducts or homocysteine. However, other endogenous substances with genotoxic properties, which are increased in ESRD, could be involved, such as the blood pressure regulating hormones angiotensin II and aldosterone or the inflammatory cytokine TNF-. This review provides an overview of genomic damage observed in ESRD patients, focuses on possible underlying causes and shows modulations of the damage by modern dialysis strategies and vitamin upplementation.
Cancer is one of the leading causes of death all over the world. Malnutrition and toxic contaminations of food with substances such as mycotoxins have been thought to account for a high percentage of cancers. However, human diet can deliver both mutagens and components that decrease the cancer risk. Genomic damage could be reduced by food components through different mechanisms such as scavenging of reactive oxygen species. In the first part of this study we tried to investigate the effects of patulin and resveratrol on DNA stability in V79 cells. Patulin is a mycotoxin, which is frequently found in spoiled apples and other fruits. The WHO has established a safety level of 50 µg/L, which is indeed not observed by all manufacturers. The acute toxicity of patulin in high concentrations is well known, however its potential carcinogenicity is still a matter of debate. Therefore we wanted to investigate further steps in the mechanism of patulin-induced genotoxicity. Patulin caused the formation of micronuclei and nucleoplasmic bridges in a dose-dependent manner. Further analysis revealed that patulin induced both kinetochore-negative and positive micronuclei. Time course of incubation indicate a new mechanism for patulin-induced nucleoplasmic bridge formation. We hypothized a mechanism via cross-linking of DNA, which was confirmed by a modified version of comet assay. Incubations of cells with patulin led to an increased number of multinucleated cells and multipolar mitoses. Cell cytometry revealed a G2 arrest by patulin, which might explain the amplification of centrosomes and patulin-induced aneuploidy. Patulin cause a dose-dependent DNA damage in comet assay which was influenced by the cellular GSH content. However, an induction of oxidative stress was just seen with higher concentrations of patulin. Levels of cellular glutathione were increased after 24 h incubation indicating an adaptive response to patulin-induced stress. There is growing interest in polyphenols such as resveratrol which have shown many positive effects on human health. The beneficial properties are partially attributed to their ability to scavenge reactive oxygen species. Co-incubation of V79 cells with patulin and 10 µM of the antioxidant resveratrol led to a slight reduction of micronucleus frequency compared to cells which were just treated with patulin. However, in higher concentrations resveratrol themselves caused the formation of micronuclei in V79 cells. Kinetochore analysis indicated only clastogenic properties for resveratrol but no disturbance of mitosis. The antioxidant properties of resveratrol were shown in ferric reducing antioxidant power (FRAP) assay. However, in cellular system resveratrol in higher concentrations revealed also prooxidative properties, as shown in 2,7-dichlordihydrofluorescein (DCF) assay. The increased level of glutathione after resveratrol treatment might reflect an adaptive response to resveratrol-induced oxidative stress. For the second part of this thesis we investigated the effects of an anthocyanin-rich grape extract on hypertensive Ren-2 rats. Ren-2 rats are an accepted genetically modified rat model for the investigation of hypertension and increased oxidative stress. We divided 23 female Ren-2 rats into three groups. One group was fed with an anthocyanin-rich Dacapo grape extract, one group was treated with the angiotensin converting enzyme (ACE) inhibitor ramipril and the third group was kept without medication during the experiment. After one week untreated group showed a clear increase in systolic and diastolic blood pressure compared to the ramipril treated rats. This was in part attenuated in the animals fed with anthocyanin-rich Dacapo grape extract. Effects on blood pressure were also reflected in an increased thirst of untreated and extract fed animals. Comet assay with cells of kidney and liver revealed a slight protective impact of Dacapo extract on DNA damage compared to the other groups. Similar results were obtained after evaluation of ɣ-H2AX-staining of kidney and heart sections. However, in the small intestine oppositional effects were seen, indicating an increased number of double strand breaks probably due to the high local concentration of polyphenols after oral ingestion. Antioxidative properties of the extract were shown in FRAP assay. However, this effect was not reflected in an increased antioxidative capacity in serum or a protective impact in the dihydroethidium (DHE) assay. The extract showed protective effects on DNA damage in comet assay and ɣ-H2AX-staining, but was not able to reduce hypertension back to the control level of ramipril treated animals. High local concentrations could also result in an increased damage of the affected tissue. Therefore, the administration of such concentrated compounds should be handled with care.
Zahlreiche experimentelle Befunde lassen vermuten, dass G-Protein gekoppelte Rezeptoren (GPCR) nach ihrer Aktivierung einer ligandenselektiven Änderung der Rezeptorkonformation unterliegen. Ziel der vorliegenden Arbeit war es dieses Phänomen am Subtyp 2 der muskarinischen Acetylcholinrezeptoren (M2 AChR) zu untersuchen. Muskarinische Acetylcholinrezeptoren (mAChR) können in fünf Subtypen (M1-M5) unterschieden werden. Durch die Beteiligung der mAChR an zahlreichen physiologischen Prozessen stellen sie wichtige Zielstrukturen pharmakologischer Therapien dar. Da die orthosterische Ligandenbindestelle (= Bindestelle des endogenen Liganden) in allen fünf Subtypen hoch konserviert ist, wird ihr pharmakologischer Nutzen derzeit allerdings durch die unselektive Rezeptormodulation und dem damit verbundenen Auftreten unerwünschter Arzneimittelwirkungen stark limitiert. Ein Ansatz zur Erzielung subtypselektiver Effekte besteht in der Verwendung allosterischer Modulatoren. Da die allosterische Bindestelle der mAChR eine geringere Sequenzhomologie aufweist, können so gezielt einzelne Subtypen der mAChR reguliert werden. Der M2 AChR stellt hinsichtlich allosterischer Modulation ein gut charakterisiertes Modellsystem dar. Für ihn wurde bereits eine Vielzahl allosterischer Liganden entwickelt. Auch bitopische Liganden, die sowohl einen allosterischen als auch einen orthosterischen Anteil enthalten, wurden für den M2 AChR bereits beschrieben. Im ersten Teil der vorliegenden Arbeit wurden verschiedene FRET-Sensoren des M2 AChR generiert und charakterisiert. Als FRET-Paar wurden das cyan fluoreszierende Protein (CFP) und der niedermolekulare fluorescein-basierte Fluorophor FlAsH (fluorescein arsenical hairpin binder) gewählt. CFP wurde in den Sensoren am Ende des C-Terminus angefügt. Die zur Markierung mit FlAsH nötige Tetracysteinsequenz wurde in verschiedenen Bereichen der dritten intrazellulären Rezeptorschleife (IL) eingebracht. Die auf diese Weise erstellten Re-zeptorsensoren trugen das Tetracysteinmotiv in der N terminalen (M2i3-N) bzw. in der C terminalen Region (M2i3-C) von IL 3. Die Charakterisierung der Rezeptorsensoren bezüglich Ligandenbindung, Gi-Protein Aktivierung und β-Arrestin2 Translokation ergab keine signifikanten Unterschiede zwischen M2i3-N, M2i3 C und M2CFP oder Wildtyp M2 AChR. Zunächst wurden sowohl unterschiedliche orthosterische, als auch allosterische Liganden hinsichtlich ihrer mittleren effektiven Konzentration und ihrer maximalen Wirkstärke an den Rezeptorsensoren untersucht. Mit Hilfe von FRET-Messungen konnte ein superago-nistisches Verhalten des orthosterischen Testliganden Iperoxo gezeigt werden. Die Eigenschaften der allosterischen Substanzen wurden durch Messung der Rezeptordeakti-vierungskinetik und des maximalen Hemmeffekts auf einen vorstimulierten Rezeptor charakterisiert. Alle allosterischen Liganden deaktivierten den vorstimulierten M2 AChR mit einer schnelleren Kinetik als Atropin. Die EC50-Werte der unterschiedlichen Substanzen waren unabhängig von der Markierungsposition im verwendeten Rezeptorsensor vergleich-bar. Ausnahmen bildeten die allosterischen Liganden JK 289, JK 338, ½ W84 und EHW 477, die liganden- und sensorabhängig unterschiedliche mittlere effektive Konzentrationen aufwie-sen. Bei der Untersuchung der Konformationsänderung des M2 AChR konnte kein liganden-selektiver Unterschied zwischen den FRET-Signalen für 19 getestete orthosterische Liganden beobachtet werden. Dies deutet darauf hin, dass alle orthosterischen Testliganden eine dem Acetylcholin (ACh) vergleichbare Änderung der M2 AChR Konformation induzier-ten. Um zu untersuchen, ob für die orthosterischen Testliganden eine Korrelation zwischen ihrer maximalen Wirkstärke hinsichtlich Rezeptoraktivierung in FRET-Experimenten und der Aktivierung nachgeschalteter Signalwege besteht, wurde die orthosterisch-vermittelte Translokation von β-Arrestin2 mit Hilfe der Konfokalmikroskopie bestimmt. Bis auf 5-Methyl-furmethiodid translozierten alle orthosterischen Liganden β-Arrestin2 in einem Ausmaß, das mit der maximalen Rezeptoraktivierung vergleichbar war. Dagegen rief 5 Methylfurmethiodid verglichen mit dem endogenen Liganden ACh zwar eine ca. halbmaximale Rezeptorakti-vierung, aber nur eine äußerst geringe β-Arrestin2 Translokation hervor. Im zweiten Teil der Arbeit wurde der Einfluss verschiedener Allostere auf eine ligandenselektive Konformationsänderung des M2 AChR untersucht. Die allosterischen Liganden JK 337 und Seminaph beeinflussten den M2i3-C Sensor signifikant stärker, als das M2i3-N Konstrukt. Dagegen zeigte EHW 477 eine stärkere Beeinflussung der Rezeptorkon-formation des M2i3-N-, als des M2i3-C Sensors. Dies erlaubt die Vermutung, dass JK 337 und Seminaph eine stärkere Bewegung unterhalb von Transmembrandomäne (TM) 6, als unterhalb von TM 5 hervorriefen. Die Ergebnisse für EHW 477 legen nahe, dass TM 5 eine größere Bewegung eingeht, als TM 6. FRET-basierte Untersuchungen der Einflüsse der allosterischen Testliganden auf nachgeschaltete Signalwege ergaben, dass sowohl die Akti-vierung des Gi Proteins, als auch die β-Arrestin2 Translokation selektiv durch einzelne allosterische Liganden beeinflusst werden. Auch ein Zusammenhang zwischen Rezeptor-aktivierung und der Regulation nachgeschalteter Signalwege war erkennbar. Allerdings waren auf Grund der Versuchsbedingungen keine quantitativen Aussagen möglich. Im Folgenden wurden die bitopischen Liganden Hybrid 1 und 2 (H 1, H 2) hinsichtlich ihres Effekts auf die Konformationsänderung des M2 AChR untersucht. Während eine Stimulation mit H 1 vergleichbare FRET-Signale an beiden Sensoren ergab, konnte mit H 2 weder am M2i3-N-, noch an M2i3-C Sensor eine FRET-Änderung detektiert werden. Um den mangeln-den Effekt der Hybridsubstanzen in FRET-mikroskopischen Untersuchungen aufzuklären, wurden verschiedene Ansätze gewählt: Mit kettenverlängerten Derivaten der Hybridsubstanzen konnte in FRET-Messungen eine Änderung des FRET-Signals detektiert werden. Die Entfernung des allosterischen Bausteins führte in FRET-Experimenten zu einer verglichen mit dem endogenen Liganden ACh etwa halbmaximalen Aktivierung beider Sensoren. Dagegen resultierte die Mutation der alloste-rischen Bindestelle in nachfolgenden FRET-Untersuchungen mit H 1 und H 2 in keiner Signaländerung des FRET-Ratio. Diese Beobachtungen führten zu der Annahme, dass die Linkerkette, die orthosterischen und allosterischen Baustein der Hybride miteinander verbindet, zu kurz war um eine gleichzeitige Bindung an die allosterische und orthosterische Bindestelle zu ermöglichen. Ein anderer Erklärungsansatz besteht darin, dass nach Bindung des Orthosters der Kanal zwischen orthosterischer und allosterischer Bindestelle durch die Konformationsänderung des Rezeptors verschlossen wird, weshalb keine dauerhafte, dualsterische Bindung der Hybridsubstanzen an den M2 AChR möglich ist. Im Rahmen der vorliegenden Arbeit ist es gelungen mittels FRET-Experimenten die Existenz einer ligandenselektiven Rezeptorkonformation des M2 AChR mit allosterischen Liganden nachzuweisen. Darüber hinaus konnte auch ein Bezug zum Auftreten einer funktionellen Selektivität mit allosterischen Liganden hergestellt werden. Die Untersuchung von 19 orthosterischen Liganden hinsichtlich ihres Einflusses auf die Rezeptorkonformation des M2 AChR ergab keinen Hinweis auf eine ligandenselektive Konformationsänderung. Die Betrachtung der orthosterisch-vermittelten Translokation von β-Arrestin2 zeigte, dass zwischen der Effizienz der orthosterischen Testliganden, den M2 AChR zu aktivieren und dem Ausmaß, in dem sie eine β Arrestin2 Translokation induzierten eine direkte Korrelation besteht. Lediglich 5-Methylfurmethiodid rief eine ungleich geringere β-Arrestin2 Translokation hervor, verglichen mit dem Ausmaß an Rezeptoraktivierung. Diese Beobachtung deutet auf die Existenz eines signaling-bias für diesen Liganden hin. Die Untersuchung der dualsterischen Liganden H 1 und 2 bezüglich ihrer Fähigkeit zur Rezeptoraktivierung ergab, dass erst durch eine Verlängerung der Linkerkette, durch die orthosterischer und alloste-rischer Baustein miteinander verbunden sind eine Konformationsänderung des M2 AChR hin zu einer aktiven Konformation erreicht werden kann. Es kann somit angenommen werden, dass in den ursprünglichen Hybridsubstanzen H 1 und H 2 eine zu kurze Linkerkette, durch die keine dualsterische Bindung der Hybride an die allosterische und orthosterische Bindestelle möglich ist, ursächlich für die mangelnde Rezeptoraktivierung des M2 AChR war.
Rtgulatory aclio11s Iaken to reduu tht risk of harmfultffects of exposure to chemieals ofltn arenot commensurDtt with the toxicologicDf risk SJsstS&ment. A numbtr of factors relating to psychology, sociology, economics Dntl politics rather than science and medicine afftct tht final decision. Wemer Lutz and colleagues illustratt the situation using tht feuktmia-indudng chtmiCJJI benzene as an examplt.
Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase cx on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzymewas 30-40% pure and form HS2 enzyme 60% with regard toprotein contents of the preparations. Form HS2 enzymewas generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptidein a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as weil as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase ß by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.
Mouse L-cells were transfected by electropenneabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin). 1be DNA concentration used was 1 l'gfml, the field strength was 10 kV fcm, the duration of the pulse was S ~s. Transfeetion yield was optimal at a temperature of 4°C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seoonds) or Ionger (3 minutes) time intervals. A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 o C. The yield of transfectants in response to 10 pulses was up to 1000 per 106 cells (using 3.3 pg DNA per cell). The inßuence of the growth phase of the cells on the transfection yield and I or the subpopulation of the mouse L--ceU line used was shown. Furthennore the clone yield depended on the DNA per ceU ratio within a very small range.
Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization
(1988)
Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 p.s) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV jcm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma celllines for use in subsequent fusion experiments.
Neoplastic cell transfonnation induced by estrogens and some other carcinogen& such as benzene appears to involve the induction of mitotic aneuploidy rather than DNA damage and point mutations. As metabolic activation may also play an important roJe in the mechanism of carcinogenesis of these nongenotoxic compounds, we have studied the Interaction of reactive quinone metabolites of various estrogens and of benzene with the major microtubular protein, tubulin, in a cell-free system. Covalent binding of the radioactively labeled metabolites to the a- and 13-subunit of tubulin was found to depend on the structure of the metabolite. When the adducted tubulins were tested in vitro for their ability to polymerize to microtubules, Inhibition of microtubule assembly was obsened in every case, although to varying extents. It is proposed that the fonnation of covalent tubulin adducts may impair the formation of mitotic spindies and thus contribute to chromosomal nondisjunction and aneuploidy induction.
In addition to hormonal activity, genetic darnage has been proposed as an important factor in oestrogen-mediated carcinogenesis. However, as short-term tests for oestrogens usually fail to show DNA mutations, lesions other than dassie nuclear DNA mutation have to be considered. Oestrogeninduced mitochondrial darnage was studied in the yeast Saccharomyces cerevisiae. Stilbene-type, but not steroidal, oestrogens were found to induce respiration-dcficient petite mutation. The effect was inversely correlated with cytotoxicity and required aromatic hydroxyl groups at the stilbene molecule. It only occurred under growth conditions and apparently was not due to the A TPase inhibitory qualities of stilbene oestrogens. Other studies have shown that petite mutation clones, which can be induced by a variety of substances, contain altered mitochondrial DNA. The mechanism of petite mutation induction might be important in tumorigenesis by also acting on nuclear DNA or facilitating carcinogenesis by disturbance of mitochondrial function.
Streptococcus pneumoniae (Pneumococcus) is one of the leading causes of childhood meningitis,pneumonia and sepsis. Despite the availability of childhood vaccination programs and antimicrobial agents, childhood pneumococcal meningitis is still a devastating illness with mortality rates among the highest of any cause of bacterial meningitis. Especially in low-income countries, where medical care is less accessible, mortality rates up to 50 % have been reported. In surviving patients, neurological sequelae, including hearing loss, focal neurological deficits and cognitive impairment, is reported in 30 to 50 %. Growing resistance of pneumococci towards conventional antibiotics emphasize the need for effective therapies and development of effective vaccines against Streptococcus pneumoniae. One major virulence factor of Streptococcus pneumoniae is the protein toxin Pneumolysin (PLY). PLY belongs to a family of structurally related toxins, the so-called cholesterol-dependent cytolysins (CDCs). Pneumolysin is produced by almost all clinical isolates of the bacterium. It is expressed during the late log phase of bacterial growth and gets released mainly through spontaneous autolysis of the bacterial cell. After binding to cholesterol in the host cell membranes, oligomerization of up to 50 toxin monomers and rearrangement of the protein structure, PLY forms large pores, leading to cell lysis in higher toxin concentrations. At sub-lytic concentrations, however, PLY mediates several other effects, such as activation of the classic complement pathway and the induction of apoptosis. First experiments with pneumococcal strains, deficient in pneumolysin, showed a reduced virulence of the organism, which emphasizes the contribution of this toxin to the course of bacterial meningitis and the urgent need for the understanding of the multiple mechanisms leading to invasive pneumococcal disease. The aim of this thesis was to shed light on the contribution of pneumolysin to the course of the disease as well as to the mental illness patients are suffering from after recovery from pneumococcal meningitis. Therefore, we firstly investigated the effects of sub-lytic pneumolysin concentrations onto primary mouse neurons, transfected with a GFP construct and imaged with the help of laser scanning confocal microscopy. We discovered two major morphological changes in the dendrites of primary mouse neurons: The formation of focal swellings along the dendrites (so-called varicosities) and the reduction of dendritic spines. To study these effects in a more complex system, closer to the in vivo situation, we established a reproducible method for acute brain slice culturing. With the help of this culturing method, we were able to discover the same morphological changes in dendrites upon challenge with sub-lytic concentrations of pneumolysin. We were able to reverse the seen alterations in dendritic structure with the help of two antagonists of the NMDA receptor, connecting the toxin´s mode of action to a non-physiological stimulation of this subtype of glutamate receptors. The loss of dendritic spines (representing the postsynapse) in our brain slice model could be verified with the help of brain slices from adult mice, suffering from pneumococcal meningitis. By immunohistochemical staining with an antibody against synapsin I, serving as a presynaptic marker, we were able to identify a reduction of synapsin I in the cortex of mice, infected with a pneumococcal strain which is capable of producing pneumolysin. The reduction of synapsin I was higher in these brain slices compared to mice infected with a pneumococcal strain which is not capable of producing pneumolysin, illustrating a clear role for the toxin in the reduction of dendritic spines. The fact that the seen effects weren´t abolished under calcium free conditions clarifies that not only the influx of calcium through the pneumolysin-pore is responsible for the alterations. These findings were further supported by calcium imaging experiments, where an inhibitor of the NMDA receptor was capable of delaying the time point, when the maximum of calcium influx upon PLY challenge was reached. Additionally, we were able to observe the dendritic beadings with the help of immunohistochemistry with an antibody against MAP2, a neuron-specific cytoskeletal protein. These observations also connect pneumolysin´s mode of action to excitotoxicity, as several studies mention the aggregation of MAP2 in dendritic beadings in response to excitotoxic stimuli. All in all, this is the first study connecting pneumolysin to excitotoxic events, which might be a novel chance to tie in other options of treatment for patients suffering from pneumococcal meningitis.
The ~fthetic oes~rog~n diethylsti~boestrol (DES) causes a dose-dependent elevation of the cytoplasuuc Ca concentratton m C6 rat ghoma cells. This Ca2+ rise is caused neither by Ca2+ influx nor ~-r release from the ~a2 + stores of the endoplasmic reticulum. Therefore it seems likely that DES mob!hzes Ca2+ from a nutochondrial source. The DES-induced Ca2+ signal is remarkably similar to the one mduced by the. tumou~ promotor ~hapsigargin. As this compound causes leakage of calcium from the endoplasmt~ rettculum tt ~ms posstble that DES induces a similar leakage from mitochondrial Ca2+ stores. It remaans to be estabhshed whether the DES-mediated rise in intracellular calcium is causally related to the tumour-promoting properties of this compound
lt is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic andjor gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endp6ints (micronucleus formation, unscheduled DNA synthesis (UDS) and cell transforrnation) were assayed under similar conditions of metabolism and dose at target in this cell system. 5-AC induces morphological transformation of SHE cells, but not UDS. Therefore, 5-AC does not seem to cause repairable DNA lesions. Furthermore, our studies revealed that 5-AC is a potent inducer of mkronuclei in the SHE system. Immunocytochemical analysis revealed that a certain percentage of these contain kinetochores indicating that 5-AC may induce both clastogenic events and numerical chromosome changes.
Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Suchchromosomes (K- Chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. Wehave analyzed primary~ immortalized and malignant marnmalian cells for the presence of kinetochore proteins and micronuclei. Our resuJts suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some Kchromosomes showed a signal. This indicates that the observed lack of kinetocbores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy.
1.2-Dioxetanes, very reactive and high energy molecules. are involved as labile intermediates in dioxygenase- activated aerobic metabolism and in physiological processes. Various toxico1ogica1 tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonucleasesensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, fonnamidopyrimidines). Pyrimidinedimersand sites ofbase loss (AP sites) which were probed by UV endonuclease and exonuclease 111 are minor lesions in this system. While the alky1-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA I 00. DNA adducts formed with an intermediary alkyJating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter. since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TAIOO and they form DNA adducts. as detected by the 212Ppostlabelling technique. Our results imply that the type of D NA darnage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA darnage by energy transfer. i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA. while radical darnage and alkylation prevail in the cellular system.
5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we and others have already reported its mutagenicity at the tk locus. Using CREST staining and C-banding studies, we showed that the induced micronuclei contained mostly chromosomal fragments although some may have contained whole chromosomes. By incorporating BrdU into the DNA of SHE cells, we determined that micronuclei were induced only when the compound was added while the cells were in S phase. Microscopically visible effects due to 5-azacytidine treatment were not observed until anaphase of the mitosis following treatment or thereafter. 5-Azacytidine did not induce micronuclei via interference with formation of the metaphase chromosome arrangement in mitosis, a common mechanism leading to aneuploidy. SupravitalUV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. In the first mitosis after 5-azacytidine treatment we observed that many cells were unable to perform anaphase separation. All of these observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA.
Tbe benzodiazepines are a class of d.rugs that are widely used in the treatment of various psychiatric disorders. One member of um ~' oxazepam, is also a common metabolite of sevmd other benzod.iazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is incol:lsb1ent, we investigated the oxazepam-induced fonnation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and LS178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all tbree ceU llnes. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were fonned in the first mitosis after treatment. Kinetochore staining (CREST -antiserum) revealed the presence of kinetochores in -SO% of the micronuclei in aU tbree ceU types. ThJs resu1t was further confinned by in situ bybridization in LS178Y cells and indicates tbe presence of wbole Chromosomes or centric fragments as weU as acentric fragments in the oxazepam-induced micronuclei. The LS178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.
This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological comequence of micronucleus formation. We compared the induction of micronuclei with mutation inductioo as measured by trißuorothymidine (TFI') resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vioblastine. AU four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained wbole cbromosomes. However, these same compounds were unable to induce TFf resistance under tbree different treatment regimes. We concluded that tbese compounds, under conditions where tbey induce primarily kinetochore positive micronuclel, were not able to induce mutations. Thus, the induction of micronuclei containing wbole chromosomes barborlog a select.able gene is not an early event leadlog to mutations in these cells.
The rate limiting step in 5-fluorouracil catabolism is catalyzed by the enzyme dihydropyrimidine dehydrogenase. Since degradation of 5-fluorouracil decreases its efficacy in chemotherapy, the inhibition of its catabolism is a promising tool. We investigated the formation of micronuclei in vitro in mouse L5178Y cells. 5-fluorouracil induced an increase in micronucleus frequency, which could significantly be enhanced by the concurrent application of 2,6-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase. The 5-fluorouracil concentration necessary to reach maximal genotoxic effects could be reduced to half in the presence of inhibitor. 2,6-Dihydroxypyridine alone and the naturally occuring enzyme substrate uracil did not induce micronucleus formation. Combined application of the chemotherapeutic agent 5-fluorouracil and an inhibitor of its could reduce side-effects by lowering the effective dose of the active drug. With this study we provide further support for the usefulness of this concept.