Browsing by discipline "Farmakologi"

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and it's activity is mediated via metabotropic GABAB - and ionotropic GABAA receptors. Receptors containing αβγ subunit combination are localized in both synaptic and extrasynaptic sites and they mediate fast synaptic phasic activity. αβδ- and α5βγ-containing receptors reside only in extrasynaptic space where they regulate tonic inhibition of the nerve cell. Disruption in tonic inhibition may cause several diseases. Drugs that selectively affect extrasynaptic GABAA receptors are believed to help treatment of diseases like sleep disorder, neuropsychiatry disorders, epilepsy, cognition impairment and recovery from stroke. Drug development of δ- selective GABAA agonists and positive modulators that enhance tonic inhibition as well as α5βγ selective inverse agonists that reduce tonic inhibition and enhance cognition are under investigation right now. Muscimol is a psychoactive molecule which activates all GABAA receptors but has higher affinity to cerebellar granule cell-specific receptor subtypes α6β2γ2 and extra synaptic α6β2δ than to the most common receptor subtype α1β2γ2. However, the binding of [3H]muscimol has produced contradictory results in former studies. Binding to membrane homogenates results to binding levels of the same magnitude in most brain regions, while in receptor autoradiography the high affinity binding is δ-subunit dependent. The affinity of muscimol to GABAAR subtypes has thus far been determined using saturation analysis, i.e. by measuring concentration-dependent binding of [3H]muscimol at equilibrium and by determining KD, the dissociation constant of the ligand. However, this value expresses the affinity of the ligand to the receptor, but does not give any information on the rate of the association and dissociation. In this master's thesis I have investigated association and dissociation rate of [3H]muscimol from recombinant GABAA receptors and from native GABAA receptors present in wild-type (WT) and δ-subunit knock-out (δKO) mice forebrain and cerebellar membranes. We concluded from binding assays that [3H]muscimol dissociates extremely slowly from δ-receptors. This explains further contradictory results: in membrane/filtration assay the washing procedure is generally much faster resulting in only low dissociation from αβγ receptors, while in the long washing procedure of receptor autoradiography only αβδ-binding is retained. The high affinity of muscimol to αβδ receptors is suggested to be due to its extremely slow dissociation from these receptors. Also association of [3H]muscimol to αβδ receptors seems to be slower, but this needs confirming studies. The exceptional binding properties of muscimol make it an interesting leading molecule in development of drugs which act via extrasynaptic GABAA receptors.

Amphetamine and its derivatives are widely used as medicines but also abused as psychostimulant drugs. The most important action of amphetamine in the central nervous system is to release dopamine to the extracellular space which leads to enhanced dopaminergic neurotransmission. Amphetamine also releases serotonin and norepinephrine by similar mechanisms and it affects indirectly other neurotransmitter systems too. It still remains partly unsolved how amphetamine exactly releases monoamines but it is known to have multiple sites of action. Amphetamine is a substrate for dopamine transporter (DAT) and it acts as a competitive inhibitor of the transporter reducing uptake of dopamine. Amphetamine enters the cell mainly through DAT and partly by diffusing through the cell membrane. The drug induces changes in DAT leading to reverse transport of dopamine from the cytoplasm into the synaptic cleft through DAT. Amphetamine is also substrate for vesicular monoamine transporter 2 (VMAT2) preventing the uptake of dopamine into storage vesicles and promoting its release from the vesicles to cytoplasm. Additionally, amphetamine inhibits monoamine oxidase (MAO), enzyme which degrades monoamines. It also enhances dopamine synthesis and according to recent studies amphetamine augments exocytotic dopamine release. Drug addiction is a chronic disorder related to structural and functional adaptive changes of neurons, called neuronal plasticity. GDNF (glial cell line-derived neurotrophic factor) is one of the many molecules regulating plasticity. It is especially important to the dopaminergic system and some investigations have suggested that it has potential as a protective agent against addiction. The aim of this study was to investigate how the overexpression of endogenic GDNF affects dopaminergic system and how it changes drug responses. A hypermorphic mouse strain (GDNFh), which is overexpressing physiological GDNF, was used. Their wild-type littermates were used as controls. Using brain microdialysis it was measured how the extracellular dopamine concentration changes in striatum and nucleus accumbens (NAcc) after amphetamine stimulation. Amphetamine was administered straight to the brain through the microdialysis probe. Microdialysis was performed on days 1 and 4, and on days 2 and 3 the mice were given amphetamine intraperitoneally. This was done to find out if the response to amphetamine changed after repeated dosing. In addition to these experiments, the biological activity of three small-molecule GDNF mimetics in intact brains was tested by microdialysis. On the first day amphetamine increased striatal dopamine output more in the heterozygous GDNFh mouse than in the wild-type mice. This stronger reaction to amphetamine may be explained by the enhanced activity of DAT in the GDNFh-het mice leading to higher intracellular amphetamine concentration. Also the striatal dopamine levels are increased in the GDNFh-het. On the fourth day no differences were detected between the genotypes. In the NAcc no significant difference was found between the genotypes. Instead in NAcc amphetamine caused a smaller increase in the dopamine output on day 4 than on day 1 in both genotypes suggesting that tolerance was developed. These results confirm that endogenic GDNF has a remarkable role in the regulation of the dopamine system and hence addiction but further investigations are needed to clarify its versatile actions. The small-molecule GDNF mimetics increased striatal dopamine output thus showing biological activity and encouraging to further investigations.

Appetite regulation is a complex process involving regulation of energy homeostasis and the rewarding nature of food. Abnormalities in appetite regulation lead to obesity and eating disorders which are challenging to treat with medicines. Especially obesity is an increasing public health problem and drug development against it is a current subject in research. Hypothalamus is the most important brain area related to appetite regulation. Also, the basal forebrain and the amygdala which are part of the reward system in the brain, contribute to the appetite regulation. There is cholinergic innervation from the basal forebrain to the amygdala and most of the cholinergic activity in the amygdala is originating from the basal forebrain. It is known that the cholinergic system inhibits appetite but there is still no research about impact of cholinergic projections between these two brain areas. Aim of this study was to find out if the cholinergic projections from the basal forebrain to the amygdala effect on appetite regulation. The study included two stereotactic surgery. In the first surgery the mice (n=14) received injections to the basal forebrain that contained genetic materials for DREADDs in AAV. DREADDs appeared in the cholinergic cells of the basal forebrain and emanated within their axons to the amygdala. In the second surgery cannulas were placed to the amygdala. CNO was injected through the cannulas to the amygdala to cause the DREADDs activate or inhibit the cholinergic cells. As a control, mice received vehicle. The feeding experiments were performed in normal conditions or after food restriction and there were either food or sugar pellets available. The pellet dispenser monitored how many pellets mice ate during the six hours after the CNO or vehicle treatment. The success of virus injections was checked after the feeding experiments by antibody dyeing. In any conditions there was no significant differences in the results due to DREADDs and treatment. Because of the small group sizes and dispersion of the results no final conclusions can be made but the additional research about this topic is needed.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. It acts upon two classes of GABA receptors: ionotropic and metabotropic. The GABAB receptor is a metabotropic G protein-coupled receptor (GPCR) that associates with the Gαi/o family of G-proteins causing inhibitory actions via the inhibition of adenylyl cyclase. Baclofen, a GABAB receptor agonist, has recently been found to be a potential treatment of alcohol addiction by suppressing the drug craving stage of the addiction cycle. However, the adverse side effects of baclofen including sedation and hypothermia, as well as its narrow therapeutic index, have limited its clinical use in the treatment. Positive allosteric modulators may be the answer to providing more selective and personalised treatments. These modulators function by enhancing or attenuating the response activated by the agonists, while having little inherent intrinsic activity. Therefore, they are less likely to cause adverse effects while also reducing the dosage of the agonistic drugs, essentially diminishing the adverse effects of the agonist. Mu-opioid receptor antagonists have been available for the treatment of alcohol and drug addiction for over 20 years. Preclinical studies have found that a continuous infusion of naltrexone has led to the increased consumption of ethanol after the drug has been metabolised out of the system. Furthermore, nalmefene, a specific and potent opioid antagonist has been developed and shown to have therapeutic advantages over naltrexone. Our study wanted to compare sex-specific brain-wide distribution of G-protein coupling after activation of GABAB receptors by baclofen and the positive allosteric modulator RacBHFF in ten RccHan®:WIST albino male and female rats, and compare species differences between the rats and five wildtype C57BL/6J male mice. Furthermore, we wanted to determine whether G-protein coupling of GABAB receptors is altered in sixty female AA (Alko, Alcohol) alcohol-preferring rats with and without various modes of treatments with naltrexone and nalmefene (7 daily injections or 7-day continuous infusion using osmotic minipumps at equipotent low and moderate doses). We did this using [35S]GTPγS autoradiography as means to measure the efficacy and potency of different concentrations of RacBHFF combined with 30 µM baclofen. Our results showed that RacBHFF does have positive allosteric activity on baclofen-stimulated GABAB receptor-mediated G-protein activation and the extent of it was regionally dependent. RacBHFF had greatest efficacy in the cerebellum, cortex and thalamus in all the samples studied. Furthermore, 30 µM baclofen alone and 10 µM RacBHFF alone are not enough to produce a statistically significant enhancement in GABAB activity in all brain regions, but RacBHFF combined with baclofen does. High concentrations of RacBHFF (100 and 300 µM) led to the complete inactivation of the receptors with G-protein coupling levels falling below non-specific binding levels, thus indicating tolerance. This was noticed in both of the sexes, as well as both of the species, indicating high system stability. Furthermore, there were no statistically significant differences between the species, and only minor regional statistically significant differences between the sexes. Comparing the regional baclofen-stimulated GABAB receptor-mediated G-protein activation between the two rat models showed that there are slight statistically significant differences, most of which are present in the nucleus accumbens, a part of the mesolimbic dopamine reward pathway. In addition, continuous 7-day infusions of a moderate dose (4 mg/kg) of nalmefene produced the greatest statistically significant enhancement of receptor stimulation by RacBHFF and baclofen when compared to the saline-treated samples. Seven daily injections of the same dose of nalmefene also produced statistically significant RacBHFF and baclofen-stimulated GABAB receptor activation in the amygdala and medial habenular nucleus. Therefore, a moderate dose of nalmefene (either as a continuous infusion or repeated injections) seems to produce the greatest overall enhancement of RacBHFF-induced stimulation of GABAB receptors. In conclusion, our results reveal that depending on the concentration of RacBHFF and the location of the receptors, RacBHFF differs in its efficacy to GABAB receptors in both wildtype and treated animal models. Furthermore, our results provide the first preclinical evidence that in the presence of opioid receptor tolerance, the GABAB receptor remains rather unaltered although the signal transductions of opioid and GABAB receptors are identical. This suggests that a combinational therapy of the GABAB receptor PAMs with a mu-opioid antagonist may potentially enhance the anti-craving, anti-alcohol-consuming habits of alcohol-addicted individuals.

Gene therapy is an experimental technique that involves inserting therapeutic genes into the target cells to treat diseases. Gene transfer can be performed by ex vivo or in vivo method. Ex vivo method means transferring the therapeutic gene in laboratory to the cells that are removed from the patient, after which the cells are returned to the patient. In the in vivo method the gene transfer is performed directly to the target tissue inside the patient's body. Gene therapy clinical trials have been carried out to treat many diseases. The majority of the clinical trials have so far been cancer trials. Nevertheless, the most promising results have been established in treating diseases that arise from mutations in a single gene, i.e. monogenic diseases. Monogenic diseases include e.g. hemophilia and heritable immunodeficiencies. The biggest challenges in the clinical trials so far have been the limited gene transfer efficiency of the currently used gene vectors, the short duration of the transgene expression and the side-effects in viral-mediated gene transfer. Nonviral gene transfer agents have so far been less efficient in vivo than the viral vectors. This is partly due to the interaction between serum components and nonviral vectors. The main purpose of this study was to investigate the effect of serum to the gene transfer efficiency of a nonviral vector polyethyleneimine PEI22K and the combination of PEI22K and cationic liposome Dosper in vitro in the SMC-cells. The potential synergistic increase in the transfection efficiency of PEI22K/Dosper combination was also studied. The secondary goal in this study was to develop an in vitro model which could be used to predict the gene transfer efficiency of gene vectors in vivo. The combination of PEI22K and Dosper resulted in a synergistic increase in the transfection efficiency in serum-free transfection. In the presence of serum the efficiency of PEI22K was higher than the efficiency of PEI22K/Dosper combination. 1-10% serum concentrations did not significantly affect PEI22K`s transfection efficiency, but dramatically decreased the efficiency of PEI22K/Dosper combination. The results suggest that PEI22K is more suitable than PEI22K/Dosper combination for in vivo gene transfer.

Tyypin 2 diabeteksen lääkehoidossa käytettäviin inkretiimimimeetteihin, GLP-1-analogeihin ja DPP-4-estäjiin, on liitetty sydän- ja verisuonisairauksien riskitekijöiltä suojaavien vaikutusten lisäksi suoria vaikutuksia kammioiden sydänlihaksiin ja -toimintaan. Kuitenkaan varmuutta GLP-1-reseptorin ilmenemisestä sydämessä ei ole oikeaa eteiskorvaketta lukuun ottamatta. Tutkimuksessa selvitetään PCR-menetelmän avulla GLP-1-reseptorin ilmenemistä sydämen eri osissa ja sydäninfarktin ja diabeteksen vaikutusta reseptoripitoisuuksiin. Tulokset näyttäisivät tukevan tutkimustietoa, jonka mukaan terveillä GLP-1-reseptoria ilmenee sydämen eteiskorvakkeissa, muttei juurikaan kammioissa. Diabetesta sairastavassa rottamallissa GLP-1-reseptorin ilmeneminen on kauttaaltaan lisääntynyt, mutta vajaatoimintainen sydän sydäninfarktin jälkeen ei ilmennä reseptoria enempää kuin terve kudos.

Review of the literature: The purpose of the review is to go through what is known about mechanisms of actions of different neurotrophic factors (GDNF, neurturin, CDNF and MANF) and how they are transported within the brain. Neurotrophic factors are endogenous and secreted proteins which have a pivotal role in the development and maintenance of neurons. They support the survival of neurons and they can help them to recover from different injuries. Due to these functions neurotrophic factors might be beneficial for the treatment of neurodegenerative disorders like Parkinson's disease. There are a great deal of studies that clearly show the neuroprotective and neurorestrorative function of GDNF and neurturin on dopaminergic neurons. They are also studied in clinical studies with Parkinson's patients but the results have been partly contradictory. The signalling route of GDNF and neurturin via RET tyrosinekinasereceptor is fairly well known but the other mechanisms of action of these factors needs to be studied further. CDNF and MANF constitute a novel, evolutionarily conserved family of neurotrophic factors. They are shown to have neuroprotective and neurorestrorative actions on dopaminergic neurons both in vitro and in vivo in a rodent model of Parkinson's disease. The mechanisms of action of CDNF and MANF are not quite clear at the moment. There are two different domains in their structure both of which are likely to carry different functions. The N-terminal domains of these proteins are close to saposins, lipid and membrane binding proteins, some of which are shown to have neurotrophic and anti-apoptotic effects. The C-terminal domain of MANF, in turn, is structurally close to the SAP-domain of Ku70-protein which binds Bax in the cytoplasm and thus inhibits apoptosis mediated by Bax. CDNF and MANF might protect neurons both via intracellular mechanisms and extracellularly acting like a secreted neurotrophic factor. CDNF and GDNF are transported retrogradially from striatum to substantia nigra. MANF, unlike the others, is transported from striatum to the frontal cortex. MANF and CDNF are shown to have better diffusion properties in the brain parenchyma than GDNF. Experimental part: We studied, by means of microdialysis, the effects of CDNF, MANF and GDNF on the dopaminergic neurotransmission of naive rats within the striatum. Neurotrophic factors (10 µg) and PBS as a negative control were injected into the left striatum in stereotaxic surgery. After this rats recovered one week before the first mircodialysis. The second mircodialysis was performed three weeks after the surgery. The samples were collected from the left striatum of freely moving rats. During the microdialysis neurotransmission was stimulated by replacing the perfusion solution with hypertonic potassium solution and with amphetamine solution. The concentration of dopamine, DOPAC, HVA and 5-HIAA was measured from the dialysate samples. In vivo TH-activity experiment was carried out for three rats in each group. NSD1015 was injected i.p.after which rats were decapitated and their striatums were dissected. The concentration of L-DOPA, dopamine and metabolites on the treated and untreated hemisphere were analyzed from the tissue samples. The amount of L-DOPA in the striatum after NSD1015-treatment indicates how active TH-enzyme is. There were no significant differences in the concentrations of dopamine and metabolites during the baseline. MANF and CDNF increased the release of dopamine from the nerve terminals compared to GDNF and PBS one week after the surgery. Three weeks after the surgery there was still significant increase in the release of dopamine in MANF group compared to GDNF group. Also the dopamine-DOPAC-turnover was increased significantly in MANF group compared to GDNF and PBS groups one week after the surgery. DOPAC/HVA -ratio was significantly smaller in GDNF group than in other groups one week after the surgery. These findings suggest that MANF potentiates dopaminergic neurotransmission most drasticly. The effects of MANF seem to last longer time than the effects of other neurotrophic factors. CDNF seems to increase the release of dopamine from the nerve terminals as well. The potentiation of dopaminergic neurotransmission could be due to increased biosynthesis of dopamine or due to the potentiation of the function of nerve terminals. In the results of the TH-activity experiment there was a trend according to which L-DOPA is synthesized less after the neurotrophic factor treatment that after the PBS treatment. This suggests that neurotrophic factors might decrease the activity of TH-enzyme.

Tutkimuksen tarkoituksena oli selvittää hevosen nivelnesteen hyaluronihapon ja proteoglyknaanin konsentraatioiden vaihtelua eri artriittitiloissa korkean erotuskyvyn nestekromatografialla (HPLC). Hyaluronihappopitoisuuden muutokset heijastavat nivelen patofysiologista tilaa. Proteoglykaanipitoisuuksien nousun avulla voidaan mahdollisesti diagnosoida rustovauriot jo ennen muiden näkyvien muutosten havaitsemista. Nykyään ainoa menetelmä rustovaurion diagnosointiin on artroskopia. Nivelnesteet analysoitiin pakastuksen jälkeen HPLC-laitteella. Analysointi oli helppoa eikä muita edeltäviä näytteiden käsittelyjä tarvittu kuin sentrifugointi ja laimennus. Tämän puolesta laite sopisi mielestäni erittäin hyvin myös rutiinidiagnostiikkaan. Hyaluronihappopitoisuudet muodostivat samankaltaisen jakauman sekä kontrolli- että potilasnäytteissä. Tämän tutkimuksen mukaan hyaluronihappopitoisuuksien perusteella ei pystytä erottamaan akuutteja eikä kroonisia artriitteja kontrollinivelistä. Proteoglyknanipitoisuuksia ei määritetty sopivan standardiaineen puutteen takia. Tyydyttiin vain toteamaan pystytäänkö ko. laitteella detektoimaan pitoisuuden vaihtelut eri artriittitiloissa. Kaikissa infektiivisissä artriiteissa (5 kpl) todettiin proteoglykaania mutta myös muutamissa muissa artriiteissa sekä jopa kontrollinivelissä. Molempien yhdisteiden konsentraatiovaihteluiden arvostelua vaikeuttaa suuret yksilökohtaiset erot jopa terveiden nivelten välillä. Jatkuvaa perustutkimusta ko. yhdisteiden parissa tarvitaan, jotta menetelmästä saadaan kliinikkojakin hyödyntävä diagnoosimenetelmä.

Lisensiaatin tutkielmani keskeisin tarkoitus on kerrata epilepsian ensisijainen lääkehoito ja yleisiä hoitokäytäntöön liittyviä asioita sekä jakaa tietoa praktiikkaa tekeville eläinlääkäreille uusista lääkevaihtoehdoista vaikeahoitoisissa epilepsia tapauksissa. Koiran idiopaattisen epilepsian ensisijaisen lääkevaihtoehdon rinnalle on tullut uusia lääkevaihtoehtoja ihmisten epilepsialääkkeistä. Kuitenkaan kaikki ihmisten epilepsialääkkeet eivät sovellu koirille käytettäväksi esimerkiksi sopimattoman farmakokinetiikan tai sivuvaikutusten takia. Lääkehoito on idiopaattisen epilepsian tärkein hoitomuoto. Suosituksena on lääkehoidon aloittaminen, mikäli kouristuskohtauksia on enemmän kuin yksi kohtaus kahden kuukauden aikana tai jos koiralla on niin sanottu pitkittynyt kohtaus (yli 5min), jolloin kyseessä voi olla status epileptikus. Ennen lääkinnän aloittamista tulisi koiralta määrittää perusverenkuva sekä elinarvot ja tutkia virtsanäyte. Tämä on erityisen tärkeää, siksi että saamme koiran yksilölliset laboratorioarvot ennen lääkityksen aloittamista. Näihin tietoihin verrataan hoidon aikana saatavia tutkimustuloksia. Lisäksi alkutiedon perusteella voidaan arvioida, mikä olisi sopivin lääkevaihtoehto ja lääkeannos kyseiselle koiralle. Koirille epilepsian hoitoon Suomessa on rekisteröity Barbivet, jonka vaikuttava-aine on fenobarbitaali. Fenobarbitaalilääkitys on suhteellisen turvallinen ja sillä saavutetaan hyvä hoitovaste 70 – 80 %:lla epileptikkokoirista. Täydellinen kohtauksettomuus saavutetaan noin 40 %:lla koirista. Koiralla, joilla kohtauksia ei saada kontrolloitua pelkällä fenobarbitaalilla tai fenobarbitaali aiheuttaa koiralle merkittäviä haittavaikutuksia, voidaan siirtyä fenobarbitaalin ja kaliumbromidi yhdistelmähoitoon. Tämä yhdistelmähoito on erittäin tehokas. Yhdistelmähoito vähentää merkittävästi elintoksisuusriskiä, sillä lääkeaineilla on eri metaboliareitit. Mikäli kohtauksia esiintyy edelleen enemmän kuin kerran 6 – 8 viikossa, suositellaan tarkempia jatkotutkimuksia. Koiralle voidaan tarvittaessa kokeilla ihmisten epilepsialääkkeitä. Eniten käytetään topiramaattia ja levetirasetaamia. Topiramaatin käyttöä koiralla ei vielä ole juurikaan kliinisesti tutkittu, mutta kliinisessä käytössä siitä on havaittu olevan hyötyä. Levetirasetaamin teho on osoitettu koirilla tehdyissä kliinisissä tutkimuksissa. Mikäli kouristuskohtaus kestää yli 5 min tai kohtauksia tulee useampi kuin yksi kohtaus vuorokaudessa, koira tulisi viedä eläinlääkäriin. Pitkittynyt kohtaus vaatii välittömästi hoitoa, sillä se voi olla hengenvaarallinen. Aktiivinen kohtaus pyritään pysäyttämään ensisijaisesti diatsepaamilla i.v.. Hoitoon kannattaa lisätä myös fenobarbitaalia i.v., sillä sen vaikutus kestää pidempään. Mikäli kohtaus edelleen jatkuu, voidaan potilaalle antaa hitaasti propofolia i.v..

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.