ࡱ> bjbjVV ͥ<<| %z z _3K___8s_ GT/""""(fB)^)DDDDDDD$tILDU -6`(|(.$6.D""qF333J.l"U"D3-D335),6"u1I 3_(0R5DF0 G5,Mz0M66 MH?n)p*3(++ n)n)n)DD3n)n)n) G----Mn)n)n)n)n)n)n)n)n)z :Title: ANTI-THROMBOXANE B2 ANTIBODIES PROTECT AGAINST ACETAMINOPHEN INDUCED LIVER INJURY IN MICE Original scientific paper Running title: TxA2 in Acute APAP Hepatotoxicity Names of authors in order of appearance: Ivan avar1, Tomislav Kelava2, Danijel Pravdi1 and Filip ulo1,2 1 Department of Physiology, School of Medicine, University of Mostar, Bosnia and Herzegovina 2 Department of Physiology, School of Medicine, University of Zagreb, Croatia Address for correspondence: Ivan avar, Department of Physiology, School of Medicine, University of Mostar, Bijeli brijeg b.b., 88000 Mostar, Bosnia and Herzegovina e-mail:  HYPERLINK "mailto:ivancavarsb@yahoo.com" ivancavarsb@yahoo.com (acceptable for the statement in the corresponding author information in the published article) Summary Prostanoids are lipid compounds that mediate variety of physiological and pathological functions in almost all body tissues and organs. Thromboxane (Tx) A2 is a powerful inducer of platelet aggregation and vasoconstriction and it shows ulcerogenic activity in the gastrointestinal tract. These observations prompted us to investigate whether TxA2 play a role in host response to toxic effect of acetaminophen (N-acetyl-p-aminophenol, APAP). Overdose or chronic use of a high dose of APAP is a major cause of acute liver failure in the western world. CBAT6T6 mice of both sexes were intoxicated with a single lethal or high sublethal dose of APAP, which was administered to animals by oral gavage. The toxicity of APAP was determined by observing the survival of mice during 48 h, by measuring concentration of alanine-aminotransferase (ALT) in plasma 20-22 h after APAP administration and by liver histology. The results have shown that anti-TxB2 antibodies (anti-TxB2) and a selective inhibitor of thromboxane synthase, benzylimidazole (BZI), were significantly hepatoprotective, while a selective thromboxane receptor (TP) antagonist, daltroban, TxB2 was slightly protective in this model of acute liver injury. A stabile metabolite of TxA2, TxB2, and a stabile agonist of TP, U-46619, had no inffluence on APAP induced liver damage. These findings indirectly support the hypothesis that TxA2 has a pathogenic role in liver toxicity induced with APAP, which was highly abrogated by administration of anti-TxB2. According to our results, this protection is mediated, at least partially, through decreased production of TxB2 by liver fragments ex vivo. Introduction Overdose or chronic use of a high dose of acetaminophen (Paracetamol, N-acetyl-p-aminophenol, APAP) represents the most prevalent cause of acute liver failure in the western world today (1-Lee, 2008). APAP, a commonly used analgesic/antipyretic drug, is considered safe at therapeutic doses and in overdose the elevated levels of the toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), are generated by hepatic cytochromes P450. (2-Mitchell, J, 1973.). Albeit the pathophysiological events that occur in the early phase of APAP toxicity have been well established, the precise biochemical mechanisms leading to cell death are not fully understood (3-James et al., 2003). It is recognized that NAPQI covalently binds with nucleophilic macromolecules such as DNA or proteins, with subsequent loss of their activity or function. Primary cellular targets have been postulated to be mitochondrial proteins as well as proteins involved in cellular ion control (4-Nelson, 1990, 3-James et al., 2003). NAPQI extensively reduces the level of hepatocellular glutathione (GSH) and this event results in a subsequent generation of reactive oxygen or nitrogen species (2-Mitchell, J, 1973; 3-James et al., 2003; 5-Jaeschke, 2000). These various oxidants promote toxicity by protein oxidation, enzyme inactivation and by damage of cell membranes via lipid peroxidation (6-Jaeschke, 2000, 7-Agarwal R, et al. 2010.). Necrosis is recognized as the mode of cell death rather than apoptosis (8-Gujral JS et al., 2002) and centrilobular hepatic necrosis is a characteristic histopathological finding in APAP overdose (2-Mitchell, J, 1973, 7-Agarwal R, et al. 2010). Prostanoids, consisting of the prostaglandins (PGs) and thromboxanes (TXs), are lipid compounds produced by sequential metabolism of membrane phospholipids (arachidonic acid) by the cyclooxygenase (COX) and specific PG/TX synthase enzymes (9-Narumiya S, 2009). Prostanoids mediate a variety of physiological and pathological functions in almost all body tissuses and organs, i.e., they regulate kidney function, platelet aggregation and neurotransmitter release, modulate function of immune system and are implicated in a broad array of diseases including inflammation, hypertension, cardiovascular disease and cancer (10-Sarah G. Harris; 11-Hata A.N., 2004). Thromboxane (TX) A2 is a potent mediator of platelet aggregation and stimulates the contractile activity of smooth muscle in blood vessels and trachea (12-Hamberg M et al., 1976; 13- HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bhagwat%20SS%22%5BAuthor%5D" Bhagwat SS et al, 1985). Increased TXA2 synthesis has been linked to cardiovascular diseases, such as angina and myocardial infarction, (14-Smith DD et al. 2010), asthma (15- Devillier, P et al., 1997) and certain ulcerative disorders in the stomach (23-Whittle BJR et al, 1981). Concerning to the role of TXA2 in liver diseases, there are some reports suggesting that TXA2 is involved in hepatorenal syndrome (16- HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Genzini%20T%22%5BAuthor%5D" Genzini T et al, 2007) and it could promote acute liver injury caused by xenobiotics (17-Quiroga J. and Prieto J, 1993). Thus, it was shown that production of TXA2 by liver homogenates ex vivo is significantly elevated in a model of liver injury induced with carbon tetrachloride (CCl4) (18-Nagai, H, 1989a), lipopolysaccharide (LPS) (19-Nagai, H, 1989b) or ethanol (20-Nanji AA, 1992b). Similarly, the overproduction of endogenous TXA2 has been founded in APAP-induced liver damage (21- ulo F, Reni M, 1995; 22- Guarner F et al, 1988). Administration of OKY-046 or OKY-1581, a selective TX synthetase inhibitors, and ONO-3708, a TXA2 receptor (TP) antagonist, highly ameliorated liver injury in above mentioned models of toxic hepatitis (18,19, 22). However, data in the study of Guarner et colleagues suggest that TXA2 inhibition per se does not reduce hepatic necrosis induced by APAP (22). Therefore, selective inhibition of the TX synthase may, besides decreasing synthesis of TXA2, increase production of PGI2 or PGE2, which protective effects have been demonstrated in various models of liver injury (17-Quiroga J. and Prieto J, 1993; 24- Yin H et al, 2007, 25-Reilly T et al., 2001,26-avar et al, 2009, 27-avar et al, 2010). Based on these data, the present studies aimed to investigate the role of exogenously applied TX and its derivatives on APAP-induced hepatotoxicity in vivo. Materials and Methods Animals CBAT6T6 mice were raised in an animal colony unit at the Department of Physiology, School of Medicine, University of Zagreb. Mice of both sexes aged 12-16 weeks and weighing 20-25 g were used in all experiments. The animal colony unit had regulated 12 h light/dark cycle and the temperature and relative humidity in the animal room were 222C and 505%, respectively. The cages were sanitized twice weekly and mice were allowed free access to tap water and standard mouse chow diet (No. 4RF21, Diet Standard, Milan, Italy). All animal protocols were approved by the Ethics Committee of the University of Zagreb, School of Medicine (Zagreb, Croatia). Chemicals and treatments of animals Pure APAP substance was a kind gift from the Belupo Pharmaceutical Company (Koprivnica, Croatia). Phenobarbitone-sodium was obtained from Kemika (Zagreb, Croatia). Since the TXA2 has a half-life of about 30 seconds, we used its stabile metabolite, TXB2, in certain experiments. TXB2 (No. 19030, Cayman Chemical, Ann Arbor, MI, USA) was supplied as a crystalline solid, dissolved in PBS (100 g/mL, pH=7.2) and thereafter injected into mice 30 min before APAP administration. Polyclonal anti-thromboxane B2 antibodies (anti-TXB2) were supplied as a lyophilized powder (No. P7291, Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in 5 mL of PBS (pH=7.2) and finally injected (40 g/kg, i.p.) into animals 3 h before APAP. Daltroban (No. D7441, Sigma-Aldrich), a selective TP receptor antagonist, was dissolved in Tris buffer (2.0 mg/mL, pH=7.4) and injected (5.0 mg/kg, i.p.) into mice 30 min before APAP. U-46619 (No. 16450, Cayman Chemical), a stable analog of the endoperoxide PGH2 and a TP receptor agonist, was purchased as a solution in methyl acetate. Organic solvent-free aqueous solution of U-46619 was prepared by evaporating stock solution under a gentle stream of nitrogen and dissolvnig the remaining substance directly in PBS (250 g/mL, pH=7.2). Thereafter, U-46619 was administered to animals (0.2 and 0.8 mg/kg, i.v.) 30 min before APAP. 1-Benzylimidazole (BZI), a selective inhibitor of thromboxane synthase, was purchased from Sigma Aldrich (No. 116416) as a crystalline solid, dissolved in PBS (1 mg/mL, pH=7.2) and injected into animals (50 mg/kg, i.p.) 2 h after APAP administration. The doses of the drugs for application in vivo were chosen from scarce data in the literature or according to the toxicity data in our preliminary experiments, in which the effects of the drugs on survival of mice and gross macroscopic changes of liver and other visceral organs were observed. Animals in control groups received appropriate vehicle. Survival of mice was followed for 48 h after APAP administration, since almost all mice either died within this period or fully recovered thereafter. Assessment and measurement of hepatotoxicity induced with APAP In order to induce hepatic cytochromes P450 (CYPs), mice were given phenobarbitone-sodium in drinking water for 7 days (0.3 g/L). Thereafter, mice were fasted overnight and APAP was given by oral gavage in a volume of 0.4 to 0.6 mL. APAP was dissolved under mild magnetic stirring in warm PBS, into which 1-2 drops of Tween 20 were added. Animals were allowed free access to food 4 h later (22-Guarner et al., 1988; 21-Renic et al., 1995). To observe the survival of the mice, APAP was administered in a dose of 250-300 mg/kg. In order to determine plasma alanine aminotransferase (ALT) concentration in plasma, as well as for histopathological evaluation of liver slices and measurement of 11-dehydro TXB2 production by liver fragments, mice were treated with high sublethal dose of APAP (150 mg/kg). Experimental and control groups of mice contained 12-13 animals (for observation of the survival) or 7-10 animals (for all other measurements). Plasma ALT activity Plasma ALT levels were determined 20-22 h after APAP administration. Mice were given 250 U heparin i.p. 15 min before bleeding and blood was collected by puncture of the medial eye angle with heparinized glass capillary tubes. After centrifugation, separated plasma was stored at -80C for 24 h before ALT determination. ALT concentrations were measured by standard laboratory techniques (21-Renic et al., 1995). Liver histology Mice were sacrificed under light ether anesthesia by cervical dislocation 20-22 h after APAP administration. Liver lobes of each animal (9-10 animals per group) were fixed in 4% buffered paraformaldehyde, dehydrated in increasing concentrations of ethanol and embedded in paraffin. Thereafter, sections of tissue were cut at 5 mm on a rotary microtome, mounted on clean glass slides and dried overnight at 37C. The sections were cleared, hydrated and stained with hematoxyllin and eosin. Microscopically, the liver damage was classified using arbitrary scale from 0 to 5 as follows: degree 0there was no damage; degree 1minimal lesions involving single to few necrotic cells; degree 2mild lesions, 10-25% necrotic cells or mild diffuse degenerative changes; degree 3moderate lesions, 25-40% necrotic or degenerative cells; degree 4marked lesions, 40-50% necrotic or degenerative cells; degree 5severe lesions, more than 50% necrotic or degenerative cells. Sections with scores higher than 2 were considered to exhibit significant liver injury (28-Silva et al., 2001). Production of TXB2 ex vivo and measurement of its concentration Mice were sacrificed 6 h after APAP administration. Blood was collected by puncture of the medial eye angle with heparinized glass capillary tubes. After centrifugation, separated plasma was stored at -80C. Samples of liver tissue, kept on ice, were minced in small fragments (1-2 mm3) in PBS. After sedimentation at unit gravity, they were washed 2 times more in fresh PBS, transferred into preweighed tubes and centrifuged at 500 g at +4C for 3 min. The sediment was quickly weighed, resuspended in Minimal Essential Medium (MEM, 5 l MEM/mg tissue) and incubated in a water bath at 37 C for 1 h. The samples were then centrifuged as above and supernatants stored at -80C until analysis. Concentration of TXB2 was determined using appropriate 11-dehydro TXB2 EIA Kit according to the manufacturers instructions (No. 514010, Cayman Chemical). Statistical analysis Data were expressed as means SEM. Differences in survival between the groups of mice were compared by chi-square test using Yates correction when indicated. Statistical comparisons between two groups were made using a Students t-test. Comparisons between multiple groups were carried out using one-way analysis of variance (ANOVA) with a post hoc test of significance between individual groups. A p-value less than 0.05 was considered statistically significant. Results Effects of TXB2 and anti-TXB2 on APAP-induced mortality and plasma ALT concentration in mice To determine the survival of animals, mice were treated with 250 mg/kg of APAP. TXB2 (2.0 mg/kg, i.p.) was given 30 min and anti-TXB2 (40 g/kg, i.p.) 3 h before APAP administration. Administration of TXB2 had no inffluence on the survival of mice (Fig. 1A, p>0.05). Administration of anti-TXB2 significantly improved the survival of animals (Fig. 1A, p<0.05). To determine the plasma ALT concentration, mice were treated as in the previous experiment, except that mice received a lower dose of APAP (150 mg/kg). Fig. 1B shows mean ALT levels (SEM) obtained in 8-10 mice per group 20-22 h after APAP administration. Treatment of animals with TXB2 increased ALT concentration, but the difference was not significant (Fig. 1B, p>0.05). Pretreatment of mice with anti-TXB2 significantly reduced ALT level (Fig. 1B, p<0.05). A  B  Fig. 1. Influence of TXB2 and anti-TXB2 on survival and plasma ALT concentration in mice with APAP-induced liver injury. A. APAP (250 mg/kg) was given by oral gavage and survival was recorded 48 h later. TXB2 (2.0 mg/kg, i.p.) was given 30 min and anti-TXB2 (40 g/kg, i.p.) 3 h before APAP administration. Vehicle was given 30 min before APAP. N = 13 mice per group.*p<0.05 in comparison to vehicle group. B. To determine the plasma ALT concentration, mice were treated as in the previous experiment, except that mice received a lower dose of APAP (150 mg/kg). Results represent mean SEM of 8-9 mice per group. **p<0.01 in comparison to vehicle group. Effect of daltroban on APAP-induced mortality and plasma ALT concentration in mice For observation of the survival of animals, mice were treated with 300 mg/kg of APAP. To measure the plasma ALT level, mice received 150 mg/kg of APAP. Daltroban (5.0 mg/kg, i.p.), a selective TP receptor antagonist, and vehicle were given 30 min before APAP administration. Pretreatment of mice with daltroban increased the survival of mice and reduced ALT level, although the differences did not reach statistical significance (Fig. 2A and B; p>0.05 for both comparisons). A  B  Fig. 2. Influence of daltroban on survival and plasma ALT concentration in mice with APAP-induced liver injury. A. APAP (300 mg/kg) was given by oral gavage and survival was recorded 48 h later. Daltroban (5.0 mg/kg, i.p.) and vehicle were given 30 min before APAP administration. N = 13 mice per group. p>0.05 in comparison to vehicle group. B. To determine the plasma ALT concentration, mice were treated as in the previous experiment, except that mice received a lower dose of APAP (150 mg/kg). Results represent mean SEM of 8 mice per group. p>0.05 in comparison to vehicle group. Effect of U-46619 on survival of mice and plasma ALT concentration To determine the survival of animals, mice received 300 mg/kg of APAP, and for measurement the plasma ALT level mice were given 150 mg/kg of APAP. U-46619 (0.2 and 0.8 mg/kg, i.p.), a selective TP receptor agonist, and vehicle were given 30 min before APAP administration. Pretreatment of mice with U-46619 did not significantly change the survival and plasma ALT level in mice with APAP-induced liver damage (Fig. 3A and B; p>0.05 for all comparisons). A  B  Fig. 3. Effect of U-46619 on survival and plasma ALT concentration in mice with APAP-induced liver injury. A. APAP (250 mg/kg) was given by oral gavage and survival was recorded 48 h later. U-46619 (0.2 and 0.8 mg/kg, i.v.) and vehicle were given 30 min before APAP administration. N = 12 mice per group.p>0.05 in comparison to vehicle group. B. To determine the plasma ALT concentration, mice were treated as in the previous experiment, except that mice received a lower dose of APAP (150 mg/kg). Results represent mean SEM of 8 mice per group. p>0.05 in comparison to vehicle group. Effect of BZI on survival of mice and plasma ALT concentration In order to determine the survival of animals and plasma ALT level, mice were treated with 300 mg/kg or 150 mg/kg of APAP, respectively. BZI (50 mg/kg, i.p.), a selective inhibitor of TX synthase, was given 2 h after APAP administration. Administration of BZI significantly improved the survival of animals (Fig. 4A, p<0.05) and decreased plasma ALT level (Fig. 4B, p<0.05). A  B  SHAPE \* MERGEFORMAT  Fig. 4. Effect of BZI on survival and plasma ALT concentration in mice with APAP-induced liver injury. A. APAP (300 mg/kg) was given by oral gavage and survival was recorded 48 h later. BZI (50 mg/kg, i.p.) and vehicle were given 2 h after APAP administration. N = 13 mice per group.p<0.01 in comparison to vehicle group. B. To determine the plasma ALT concentration, mice were treated as in the previous experiment, except that mice received a lower dose of APAP (150 mg/kg). Results represent mean SEM of 10 mice per group. p<0.001 in comparison to vehicle group. Liver histology Macroscopically, the whole liver surface of some APAP treated mice had a mottled appearance; dark red hemorrhagic-necrotic spots were regularly scattered on the yellowish background. Microscopically, the liver damage was graduated using arbitrary scale from 0 to 5 as described in Materials and methods (Fig. 5). The severity of necrosis was quite variable both between animals and also within different parts of the same liver. However, anti-TXB2 or BZI significantly decreased the number and size of necrotic foci in the liver, which could be easily seen by macroscopic observation and on histological analysis. Macroscopic and microscopic damages of the liver parenchyma appeared more pronounced in mice injected with TXB2, although the differences did not reach statistical significance (Table 1).  Fig. 5. Histopathological changes in livers from normal and APAP-intoxicated mice. Livers were collected 20-22 h after APAP administration (150 mg/kg). Graduation of liver damage was determined according to the arbitrary scale as follows: degree 0 (A), degree 1 (B), degree 2 (C), degree 3 (D), degree 4 (E) and degree 5 (F). Descriptions of each degree are explained in Materials and methods. Sections were stained with hematoxyllin and eosin (original magnification, x 10). Table 1. Effects of TxB2, anti-TxB2 and BZI on APAP-induced liver injury Stupanj histopatoloakog oateenjabTreatmenta012345Stupanj>2cVehicle + APAP0211315/8TxB2 + APAP0222204/8Anti-TxB2 + APAP 232010 1/8*APAP + BZI1311113/8 Mice were sacrificed and livers were collected 20-22 h after mice received APAP (150 mg/kg). a: anti-TxB2 (40 g/kg, i.p.) was given 3 h, while TxB2 (2.0 mg/kg, i.p.) and vehicle were given 30 min before APAP administration. BZI (50 mg/kg, i.p.) was given 2 h after APAP. b: Histopathological scores were determined and graded by intensity of hepatocellular necrosis from 0 to 5 as described in Materials and methods. c: Scores greater than 2 were considered as significant necrosis. N = 8 mice per group. *Statistically significant in comparison to vehicle group (p<0.05). Effect of anti-TXB2 on the production of TXB2 ex vivo TXB2 production was determined in plasma and supernatants of incubated liver fragments taken from normal (non-treated) mice and mice treated with anti-TXB2 (40 g/kg, i.p.) or vehicle 2 h after APAP administration. In comparison to normal mice, treatment with APAP alone (vehicle group) significantly increased production of TXB2, while treatment with anti-TXB2 reduced that increase in TXB2 production (Fig. 6, p<0.05 for both comparisons).  Fig. 6. Effect of anti-TXB2 on TXB2 production by the plasma and liver fragments. Anti-TXB2 (40 g/kg, i.p.) and vehicle were given 3 h before APAP administration (150 mg/kg). Plasma and liver samples were taken 6 h after APAP administration. TXB2 concentration was determined in plasma and supernatants obtained after 1 h incubation of liver fragments. Results represent mean SEM of 6 mice per group. #p<0.05 in comparison to normal mice. *p<0.05 in comparison to group which received anti-TXB2. Discussion Intoxication with APAP is a major cause of acute liver failure in the western world (1-Lee, 2008) and from that reason we used it as a model of drug-induced liver injury for examination the influence of anti-TXB2 and derivatives of TXA2 on drug toxicity mechanisms. The presented results clearly demonstrate that tretament of mice with anti-TXB2 improved the survival of mice and alleviated hepatic damage, as assessed by plasma ALT concentration and liver histology. As far as we know, this is the first time that anti-TXB2 has been used in vivo in a model of experimental liver damage induced by a noxious agent. Nevertheless, the mechanism of its protective action is not known and our results indicate that it could be expressed through the inhibition of production of TXB2 by liver homogenates. This indirectly points to TXB2 as an endogenously produced hepatotoxic agent, which is supported by our observation that APAP alone increases synthesis of TXB2 in the liver. Thus, it was shown that APAP also increases hepatic synthesis of PGE2 and PGI2 (22, 26, 27), each of which has been shown to have a significant hepatoprotective effect (22, 24-27). The elevated levels of TXB2 have been founded in liver and systemic circulation after different types of hepatic injury such as endotoxemia, hepatic ischemia-reperfusion, hepatectomy, liver transplantation, hepatic cirrhosis, and after liver injury induced by ethanol and CCl4 (18-Nagai, H, 1989a; 20-Nanji AA, 1997b; 29-Yokoyama Y et al, 2005; 30- Katagiri H, et al 2004; 31-Shimada M et al, 1994; Shirabe K et al, 1996). Due to a unstabile nature and very short half-life of TXA2, its stabile metabolite, TXB2, was used in our investigations. Although TXB2 is considered as a biologically inactive metabolite of TXA2, it has been improved its biological activity in several studies (40-Pascual J et al., 1988;  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hrushka%20NH%22%5BAuthor%5D" Hrushka NH, 2006). In our experiments, treatment of mice with TxB2 had no significant influence on the survival, plasma ALT level and liver histology. In order to detrmine its biological activity, TXB2, in a various doses, didnt cause elevation in platelet aggregation induced with ADP or arachidonic acid in mice plasma (data not shown). Hepatoprotective effect of anti-TXB2 remains unclear, however, it could be expressed through its cross-reactivity with TXA2 and TXB2 by consequent inactivation of these compounds. In the present experiments, treatment of mice with daltroban, a selective antagonist of TP receptor, alleviated the liver damage induced by APAP as shown by the decrease in mortality of animals and plasma ALT level. Numerous animal studies have shown that other specific TP receptor antagonists protect the liver from injury after toxic dose of ethanol (33-Nanji AA et al.) and CCl4 (18-Nagai), endotoxemia (19-Nagai, 30-Katagiri), warm ischemia (32-Shirabe et all, 1996) ischemia-reperfusiuon (29-Yokoyama Y et al, 2005; 35- HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Suehiro%20T%22%5BAuthor%5D" Suehiro T et al, 1994) etc. Except the inhibition of TP receptors, another important mechanism to protect hepatic tissue is increase in the production of PGI2 in the liver (34-Hanazaki K et. All, 1994). On the other side, administration of a stabile analog of TXA2, U-46619, to normal mice or animals which received APAP had no influence at all on the parameters of liver injury. Previously, it was demonstrated that U-46619, when was injected i.v. into the mice, caused clear elevation of serum ALT and AST levels and aggravated the histopathological score of liver necrosis (18- HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nagai%20H%22%5BAuthor%5D" Nagai H. Et al.). We are not able to offer approppriate explanation for biological inefficacy of U- 46619, except that different mice strain was used in our investigations. Our results clearly show that BZI, a a selective inhibitor of TX synthase, when it was given to mice 2 h after APAP significantly improved the survival and decreased plasma ALT level of treated animals. Guarner et al. demonstrated that BZI has a strong protective effect on APAP overdose in mice, but only when it was given to animals 30 min before lethal dose of APAP. Furthermore, the same authors showed that BZI interfered with hepatic drug metabolism, as assessed by prolongation of the pentobarbitone sleeping time (22-Guarner et al., 1988). Data in this study also demonstrated that inhibition of TXA2 production per se is not sufficient for conferring protection against APAP-induced liver injury; concomitant elevation of concentration of endogenously produced or exogenously applied PGI2 and PGE2 was essential for the protection of intoxicated animals (22-Guarner et al., 1988; 26-avar et al.; 27-avar et al.). Since the generation of NAPQI is almost completed in the first 2 h after an APAP overdose, we excluded the possibility that protective action of BZI is mostly due to its interference with APAP metabolism. The mechanism of its protective action is most probably due to the inhibition of TXA2 synthesis, and besides, in our experiments the dose of BZI was much higher in comparison to that used in the previously described study. In addition, there are also evdences that decrease in TXA2 synthesis by inhibition of TX-synthase or COX-2 has beneficial effect on liver injury induced by various toxic agents other than APAP, such are ethanol (33-Nanji AA et al.) and CCl4 (18-Nagai), endotoxemia (19-Nagai, 30-Katagiri, 2004, 36-Ito Y et al-2003), ischemia-reperfusiuon (29-Yokoyama Y et al, 2005; 35- HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Suehiro%20T%22%5BAuthor%5D" Suehiro T et al, 1994) etc. The results reported here indicate that TXA2 plays an important role in the onset and development of experimental liver diseases in mice. Among the eicosanoids, TXA2 has been shown to promote inflammatory process in the liver, hepatic microcirculatory disturbance through its vasoconstrictive effect in the portal venous system and leukocyte adhesion in the sinusoids, all of which lead to hepatic tissue injury (29-Yokoyama Y et al, 2005). Studies have shown that, in endotoxemia (LPS)-induced liver damage in mice, enhanced release of TXA2 from the liver is responsible for the high production of a representative proinflammatory cytokine, tumor necrosis factor alpha (TNF-). In addition, TXA2 is associated with the increased expression of intercellular adhesion molecule 1 (ICAM) in the hepatic microvasculature, which facilitates leukocyte adhesion and contributes to the hepatic inflammatory process (30-Katagiri, 2004). Several studies have shown that TXA2 induces the release of endothelin, which contributes to the liver inujry through its potent vasoconstrictive action (29-Yokoyama Y et al, 2005; 37- HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Yokoyama%20I%22%5BAuthor%5D" Yokoyama I et al., 1996). Furthermore, TXA2 upregulates other vasoconstrictors, such is angiotenzin II, and downregulates vasodilators, such are PGI2 and NO, and these effects further exacerbate the vasoconstrictive effects of TXA2 (29-Yokoyama Y et al, 2005). In an animal model of ischemia-reperfusion liver injury (ex vivo) has been shown increase in level of lipid peorxide and decrease in concentration of antioxidants, glutathione and alpha-tocopherol (38-Haba Y et al, 1995). In this regard, administration of different COX inhibitors, specific TX synthase inhibitors and specific TP receptor antagonists has been shown to abrogate these negative effects of TXA2 and protect from severe hepatic injury elicited with, above mentioned, hepatic stresses (29-Yokoyama Y, 2005; 33-38). It seems that PGI2/TXA2 ratio is critical for maintenance of the hepatic microcirculation, since its decrease has been shown in several models of liver injury (32-Shirabe et all, 1996; 38-Haba Y et al, 1995; 21- ulo F.,et al 1995; 31-Shimada M et al, 1994). Prostaglandin I2 antagonizes the effects of TXA2 by inducing vasodilation and inhibiting platelet aggregation (29-Yokoyama Y, 2005). Thus, exogenously apllied PGI2 or its stabile agonists significantly increased PGI2/TXA2 ratio and improved the outcome of liver injury induced by APAP, as well as by other noxious agents, in vivo and in vitro (17-Quiroga J, 1993; 21- ulo F.,et al 1995; 22-Guarner et al., 1988; 26-avar et al 2010.; 27-avar et al. 2009; 24-Yin H.,, 2007). 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Eur Surg Res. 1995;27:100-110.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Iwasaki%20W%22%5BAuthor%5D" Iwasaki W,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kume%20M%22%5BAuthor%5D" Kume M,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kudo%20K%22%5BAuthor%5D" Kudo K,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Uchinami%20H%22%5BAuthor%5D" Uchinami H,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kikuchi%20I%22%5BAuthor%5D" Kikuchi I,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nakagawa%20Y%22%5BAuthor%5D" Nakagawa Y,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Yoshioka%20M%22%5BAuthor%5D" Yoshioka M,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamamoto%20Y%22%5BAuthor%5D" Yamamoto Y. Changes in the fatty acid composition of the liver with the administration of N-3 polyunsaturated fatty acids and the effects on warm ischemia/reperfusion injury in the rat liver.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/19543146" \l "#" \o "Shock (Augusta, Ga.)." Shock. 2010 Mar;33(3):306-14.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Pascual%20J%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract" Pascual J,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Sterin-Borda%20L%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract" Sterin-Borda L,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Wald%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract" Wald M,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Borda%20ES%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract" Borda ES. TXB2: cardiostimulant effect that involves beta-adrenoceptor and Na+ + K+-ATPase activity.  HYPERLINK "javascript:AL_get(this,%20'jour',%20'Prostaglandins%20Leukot%20Essent%20Fatty%20Acids.');" \o "Prostaglandins, leukotrienes, and essential fatty acids." Prostaglandins Leukot Essent Fatty Acids. 1988 Jul;33(1):53-9.  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hrushka%20NH%22%5BAuthor%5D" Hrushka NH,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Korni%C4%ADchuk%20HM%22%5BAuthor%5D" Korni-chuk HM,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Makohon%20NV%22%5BAuthor%5D" Makohon NV,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Aleksieieva%20IM%22%5BAuthor%5D" Aleksieieva IM. Effect of thromboxane B2 on morphological features of apoptosis in cultured rat hepatocytes exposed to hepatotropic substances.  HYPERLINK "javascript:AL_get(this,%20'jour',%20'Fiziol%20Zh.');" \o "Fiziolohichny- zhurnal (Kiev, Ukraine : 1994)." Fiziol Zh. 2006;52(5):34-40. between development of experimental alcoholic liver injury and plasma levels of endotoxin a selective inhibitor of thromboxane synthase After an overdose of APAP, elevated levels of the toxic NAPQI metabolite are generated, which can extensively deplete hepatocellular GSH, covalently bind to cellular macromolecules with sequent modification of its function, and finally cause hepatocyte death. The precise biochemical mechanism of cell necrosis is not fully understood. However, it is generally recognized that there is simultaneous involvement of covalent binding, lipid peroxidation and oxidative stress, each contributing to hepatocellular damage6,7. In overdose, the analgesic/antipyretic acetaminophen produces centrilobular hepatic necrosis (Mitchell et al., 1973a).Cytochrome P450 metabolism to N-acetyl-p-benzoquinone imine (NAPQI) is a critical step. NAPQI reacts with hepatic glutathione (GSH) leading to its depletion by as much as 90% (Mitchell et al., 1973b). Additionally NAPQI covalently binds to proteins as acetaminophen-cysteine adducts (Cohen et al.,1997). Immunochemical studies indicate that the cellular site of covalent binding correlates with the toxicity (Hart et al., 1995; Roberts et al., 1991). Whereas depletion of intracellular lutathione (GSH) by the active APAP metabolite, Nacetylbenzoquinoneimine (NAPQI), is an early event leading to oxidative stress, reactive oxygen and nitrogen species generated by hepatic nonparenchymal cells and infiltrating phagocytes contribute to the persistence of this response (Gardner et al., 1998; Laskin and Gardner, 2003; Michael et al., 1999). In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by initial hepatic metabolism to N-acetyl-p-benzoquinone imine (NAPQI). Following low doses NAPQI is efficiently detoxified by glutathione (GSH). However, in overdose GSH is depleted, NAPQI covalently binds to proteins as APAP adducts, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. It was shown that acetaminophen is metabolically activated by cytochrome P450 to form a reactive metabolite that covalently binds to protein (Mitchell et al., 1973). The reactive metabolite was found to be N-acetyl-p-benzoquinone imine (NAPQI1), which is formed by a direct two-electron oxidation (Dahlin dddddXeYebeceeefeffffffffffffYgZgbgcgegggfhghhhhhhhii i igihijjjjjjjjjjkkkkllmmmmmmLnϽhB9B*CJaJph hjhW.jh[uh[uU h[uh[uh[ujh[uUhdl hdlhdl#jhdl0J!UmH nHsH tHBLnynznpppp q!qqqr"rrrrrrrDtEtttttuvvvv!w߁ͽͽͽͽͽͽ~vrvrvrvrh_%jh_%UhKh)S*UhKhx6S*]hKhx6B*S*]ph3fhKhxB*S*ph3fhKhxB*S*phhKhx6B* S*]phhKhxB* S*ph$hKhxB*OJQJS*^Jph# hKhxS*hKhx0J S*)pprrsCtDtuuvwށ߁ 7$8$H$gdc 7$8$H$gdx dh7$8$H$gdxet al., 1984). After a toxic dose of acetaminophen, total hepatic GSH is depleted by as much as 90%, and as a result, the metabolit covalently binds to cysteine groups on protein, forming acetaminophen- protein adducts (Mitchell et al., 1973). The covalent binding of the reactive metabolite Nacetyl-para-benzoquinone imine (NAPQI) to sulphydryl groups of hepatocyte proteins has been identified as the initiating mechanism eventually leading to hepatocellular necrosis (3,4).     Ovdje bi mi trebala novija referenca o djelovanju TxB2 in vivo; znam da ste vi imali nekoliko radova o biloakoj aktivnosti TxB2, jai h nikako ne mogu pronai na PubMedu Ovo u na samom kraju srediti Umjesto njega ide referenca Hanazaki(34) Ovo umjesto 32 ***  EMBED E.0lnƒºhKh)S*hj_ h44Uj!>P h44CJUVaJUjh44Uh_%h<h44mHsH h[uh44h44jh440J!U.l 7$8$H$gdcgdzR xcel.Chart.8 \s  6&P 1h:pN*Q. 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A        "       (   (  1"< "  AChart4lHSheet1GSheet2Sheet7Sheet6Sheet5)Sheet9LSheet81qSheet4Sheet3 >  "`, )   3 A@@   AB broj ~ivih broj mrtvihpostotak pre~ivjelihDatum: 06.06.2007RPokus: I-1 (TXB2-tromboksan B2, anti-TXB2, APAP) - jetrene transaminaze: ALT, AST'praenje pre~ivljenja: 2 dana (48 sati)A = F.O. + APAPB = TXB2 + APAPskupina oznaka AST (U/l) ALT (U/l) A A,/1 A,/2 A,/3 A,/4 A,/5 A,/6 A,/7 A,/8 A,/9 B B/1 B,/2 B,/3 B,/4 B,/5 B,/6 B,/7 B,/8 B,/9 B,/10 C C,/1 C,/2 C/3 C/4 C/5 C/6 C/7 C/8 C/9 C/10 C/11 Stupanj hemolizesrednja vrijednostgrupaASTALT+ako se izbaci najvea i najmanja vrijednost@Pokus: I-2 (TXB2-tromboksan B2, anti-TXB2, APAP) - pre~ivljenjeDatum: 05.09.2007"Miaevi: CBA/IRB, 39 ~enki, e" 3 mj."Miaevi: CBA/IRB, 30 ~enki, e" 3 mj.MDoze: APAP=300 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miausati nakon APAP br. ~ivih miaevaB= TXB2 + APAP.& & & & & & ..7& & & & & & & & & & & .7 & & 5& & & & & & & .MDoze: APAP=150 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miauSkup. ASkup. Bvrijeme nakon inj. APAP (sati)Skup.Cbroj pre~ivjelih ~ivotinja$Miaevi: CBA/ T6T6, 32 ~enki, e" 3 mj.DD/1 D/2D/3D/4D/5D/6Datum: 08.03.2008SKUPINE (N=8): jedanaestdevet sedamdesetosamsedamSKUPINE (N=12):SKUPINE (N=9-11):SKUPINE: (N=13)Z Doze: TXB2=0,2 mg/kg, otopljen u DMSO (10 mg/mL), i.p. 30 min. prije APAP, u V=0.2 mL/miaud  anti-TXB2-liofilizirana protutijela otopljena u 5 ml PBS, i.p. 2 sata prije APAP, u V=0,2 mL/miau & A= F.O. + APAP $Ovdje napraviti graf za pre~ivljenje.Napomena: Ovdje napraviti graf za pre~ivljenje .........STDEVSEM; crveni brojevi su izba eni iz statistike da se smanji STDEV*Napomena: ovdje napraviti graf samo za ALTC = anti-TxB2 + APAPDatum: 23.09.2009%Miaevi: CBAT6/T6, 24 mu~jaka, e" 3 mj.$Doze: APAP=160 mg/kg i.g. u V=0,5 mLO Doze: anti-TXB2 (40,0 g/kg), otopljen u F.O., 2 h prije APAP, u V=0.2 mL/miauSKUPINE (N=6):&A = F.O. + APAP (homogenati jetre) (B = anti-TXB2 + APAP (homogenati jetre) C = F.O. + APAP (plazma) D = anti-TXB2 + APAP (plazma)Skup.BSkup.D0average x razrjeenje (x6 za A i B; x3 za C i D)Datum: 31.08.2009'Miaevi: C57Black/6, 24 mu~jaka, e" 3 mj.$Doze: APAP=375 mg/kg i.g. u V=0,5 mLA = APAP + F.O. D = APAP + BZI (2 h nakon APAP)vrijeme proteklo od inj. APAP Skup.ASKUPINE (N=13):%koncentracija 11-dehidro-TXB2 (pg/mL)Datum: 30.09.2009'Miaevi: C57Black/6, 34 mu~jaka, e" 3 mj.$Doze: APAP=200 mg/kg i.g. u V=0,5 mLG Doze: BZI= 50,0 mg/kg, otopljen u F.O., 2 h nakon APAP, u V=0.2 mL/miauSKUPINE (N=17):A = APAP + F.O. &B = APAP (200 mg/kg) + BZI (50 mg/kg) A/1 A/2 GRUPA STDVA/3 A/4 A/5 A/6 A/7A/8 A/9A/10 A/11 A/12 B/2B/3 B/4 B/5B/6 B/7B/8 B/9 B/10 B/11 B/12 B/13/ crveni brojevi su izba eni radi smanjenja STDEV %Napomena: ovdje napraviti graf za ALTm Doze: BZI= 50,0 mg/kg, otopljen u F.O., i.p. 30 min. prije (nije prikazano) i 2 h nakon APAP, u V=0.2 mL/miauD = U-46619 (sam)iKomentar: Ovdje je o ito da U-46619 nema nikakvog u inka, pa ne treba niata ra unati- samo napraviti graf*Pre~ivljenje: kroz 48 h od primjene APAP-aMDoze: APAP=250 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miau1B = U-46619 0,2 mg/kg (30 min. prije APAP) + APAP1C =U-46619 0,8 mg/kg (30 min. prije APAP) + APAPDoze: U-46619 0,2 mg/kg i 0,8 mg/kg, isparen pod nje~nom strujom N2, otopljen u PBS-u (2 mg/mL), i.v. 30 min. prije i 2 sata nakon APAP, u V=0.2 mL/miau1C = U-46619 0,8 mg/kg (30 min. prije APAP) + APAP\Pokus: I-8 (Daltroban-kemijski antagonist TXA2r) - odreivanje ALT-a (20-22 h nakon APAP-a)Datum: 03.06.2009Doze: Daltroban 5,0 mg/kg, otopljen u TRISu (5 mg/3mL), potom razrijeen u PBS-u i dat i.p. 30 min. prije APAP, u V=0.2 mL/miau)B = Daltroban (30 min. prije APAP) + APAP;Average (kad se izbaci najvea vrijednost iz svake skupine)( crveni brojevi su izba eni iz statistike %Komentar: Ovdje napraviti graf za ALT&Miaevi: CBA/ T6T6, 16 mu~jaka, e" 3 mj.LPokus: I-9 (Daltroban-kemijski antagonist TXA2r) - odreivanje pre~ivljenjaDatum: 06.06.2009&Miaevi: CBA/ T6T6, 26 mu~jaka, e" 3 mj. pre~ivljenjeENapomena: Nije potrebno ra unati, jer nema statisti ki zna .razlike; C= anti-TXB2 + APAPWPokus I-7: Benzylimidazole (BZI) - kem. antagonist TXA2 sintetaze- ALT-"time response"QPokus I-6: Benzylimidazole (BZI) - kem. antagonist TXA2 sintetaze-"pre~ivljenje"r Pokus I-5: anti-TXB2 protutijela - odreivanje "ex vivo" produkcije 11-dehidro-TXB2 u homogenatima jetre i plazmi19UWPokus: I-4 (U-46619-stabilni agonist TXA2r) - odreivanje ALT-a (20-22 h nakon APAP-a)\Pokus: I-3 (U-46619-stabilni agonist TXA2r) - odreivanje pre~ivljenja miaeva-dose responseC = anti-TXB2 + APAP Naziv grupa: B-TXB2 + APAPC-anti-TXB2 + APAPUkoliko ti nazivi za za X-osu budu predugi, izbaci +APAP, tako da ostanu samo kemikalije (v. kao i za PGE2) - ovo vrijedi i za ostale grafoveA-Vehikulum + APAP  VehikulumU-46619 (0.2 mg/kg)U-46619 (0.8 mg/kg)! U-46619 (0.2 mg/kg)! U-46619 (0.8 mg/kg)3D-ovo ne treba ii na g<raf, spomenut u to u tekstu|Napomena: ovdje napraviti graf (usporeuju se skupine A i B te C i D); ato se ti e naziva osi napravi kako ti je najzgodnije<Napomena: Ovdje napraviti graf za pre~ivljenje; ovo e biti /Slika 13A, udru~uje se sa prethodnom slikom 13B$Slika 14A, udru~uje se sa slikom 14B/Slika 14B, udru~uje se sa prethodnom slikom 14A.Slika 15A, udru~uje se sa sljedeom slikom 15B.Slika15B, udru~uje se sa prethodnom slikom 15A/Slika 16A, udru~uje se sa prethodnom slikom 16B.Slika 16B, udru~uje se sa sljedeom slikom 16ASlika 17acsig 0.01bcsig 0.05 TXB2 + APAPanti-TXB2 + APAPAPAP + vehikulum APAP + BZI sig 0.001#bili krivi stdev i sem u obje grupeDaltroban + APAP NS p=0.07!bile pogreane sd i SEM za grupu CKomentar: Samo dodati po 1 stupac lijevo koji bi se odnosio na normalne miaeve u podioke plazma i homogenati. Naime, konc. TXB2 u plazmi i homogenatu normalnih miaeva je 1-2 pg/mL. Ne trebaa stavljati nikakve zvjezdice niti ata drugo, nego samo dodati po 1 mini stupac za normalne miaeve Komentar: 9Ovo e biti slika 13B i nju treba udru~iti sa slikom 13A Slika 13B Slika 13AAnti-TXB2 + APAPVehicle + APAPPlasmaLiver fragmentsNormalU-46619 (0.2 mg/kg) + APAPU-46619 (0.8 mg/kg) + APAP" p&bLD.V@! pZF"0 U$? o%Y ]&G'8("p(Z(m+W/26{7:_<Y=T@  "`u t??t3` / D` / E` / Fc3d23 M NM4 3QQ ;Q ;Q3_ O   MM<4E4 3QQ ; QQ3_ O   MM<4J[x@4 3QQ ; QQ3_ O   MM<4J[x@4D $% MP(3O&DQ4$% MP(3O&EQ4FAu , 3O-*3 b#M43*@@@#M4% _MZ3O7&FQ  ALT (U/L)'43" 444e)APAP + vehikulum APAP + BZIeE]t@x@x@tE]k@G@G@e>    dMbP?_*+%M EPSON AL-M2000 Advanced2  4dXXA4 210 x 297 mm XX,4 4 dd @XX*DLLName32=EPOBF7AC.DLLQ`C:\WINDOWS\system32\" dX??U-                              0 0 0           0 0 0 0RPokus: I-1 (TXB2-tromboksan B2, anti-TXB2, APAP) - jetrene transaminaze: ALT, AST Datum: 06.06.2007 M"Miaevi: CBA/IRB, 30 ~enki, e" 3 mj. MDoze: APAP=150 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miau ZDoze: TXB2=0,2 mg/kg, otopljen u DMSO (10 mg/mL), i.p. 30 min. prije APAP, u V=0.2 mL/miau d anti-TXB2-liofilizirana protutijela otopljena u 5 ml PBS, i.p. 2 sata prije APAP, u V=0,2 mL/miau & SKUPINE (N=9-11):  A = F.O. + APAP   B = TXB2 + APAP   C = anti-TXB2 + APAP   2;crveni brojevi su izba eni iz statistike da se smanji STDEV   skupina  oznaka   AST (U/l)  U ALT (U/l)  Stupanj hemolize   !A "A,/1 #`@#С@#  !A "A,/2 $@#\@ #_u+ako se izbaci najvea i najmanja vrijednost vvv  !A "A,/3 $n@N@z@N   !A "A,/4 $@#@ #wsrednja vrijednostw   !A (A,/5 #`@#L@ # Naziv grupa: AST ALT STDEV  SEMK !bile pogreane sd i SEM za grupu C !A (A,/6 #<@#@ #%Vehicle + APAP~ %b@,%ȿ@%%B, ﳺ]@%%B ~  YA  )A *A,/7 +\@+@ +& TXB2 + APAP~ &@1&@$%% B1 2O~7K@$%% B ~  # A  )A ,A,/8 +@O@ O3anti-TXB2 + APAP~ 3@13qq̔@$!%#%%'+B t@@@   )A ,A,/9 ~ +"@- .........+  .B  /B/1 #0@#x@#3T*Napomena: ovdje napraviti graf samo za ALT    .B 0B,/2 $.@N@N{T9Ovo e biti slika 13B i nju treba udru~iti sa slikom 13A    .B 0B,/3 1@1,@1TUkoliko ti nazivi za za X-osu budu predugi, izbaci +APAP, tako da ostanu samo kemikalije (v. kao i za PGE2) - ovo vrijedi i za ostale grafove    .B 0B,/4 #a@#d@ ##88@ %B  .B 0B,/5 $D@NN@ N(b@$%B  4B 5B,/6 +@+@+1 {v@$!%#%%'+B    4B 6B,/7 +*@+@ + ALT  4B 6B,/8 +k@+ @ + acsig 0.01  4B 6B,/9 +$@+@ + bcsig 0.05  4B  6B,/10  +@+@ +BX:m:88=MQx_i{{! 0"  #  $  %  &  '  (  )  * 0+ 0,  !7C !8C,/1 !#a@#f@!# "7C "9C,/2 "$@N`@"N #7C  #9C/3 ##@#@ ###T Slika 13B $7C  $9C/4 $#@#@$# %7C  %9C/5 %#@# @%# &7C  &9C/6 &$T@NN@&N '7C  '9C/7 '#i@#i@'# (7C  (9C/8 (#@#`@(# ):C  );C/9 )+V@+Y@)+ *:C *<C/10 *+r@+@z@*+ +:C +<C/11 ++v@+`@++,:II\HHHHHHII(  F^  # 2 7] ``Zph  0A@?]` ph  0A@? ]` n^  # $d@1] `|t  <NMM?]   M"HP LaserJet P1005D dXXHP LaserJet P1005  ld'4@ @ dd@ " dXX??3` / $` / &` / '?pп3d23 M NM4 3QQ ;Q ;Q3_ O NM  MM<4E4 3QQ ; QQ3_ O   MM<4J[Hz@4 3QQ ; QQ3_ O   MM<4J[Hz@4D $% MP(3O&&Q4$% MP(3O&'Q4FAl n 3O2F3 b#M43*p@@i@#M4% [$MZ3O;&$Q  ALT (U/L)'43" 444eeHz@Hz@Q@Q@@@@@e   6$@5 55  7   dMbP?_*+%M EPSON AL-M2000 Advanced2  4dXXA4 210 x 297 mm XX,4 4 dd @XX*DLLName32=EPOBF7AC.DLLQ`C:\WINDOWS\system32\" dXX??U(                               @Pokus: I-2 (TXB2-tromboksan B2, anti-TXB2, APAP) - pre~ivljenjeDatum: 05.09.2007M"Miaevi: CBA/IRB, 39 ~enki, e" 3 mj.MDoze: APAP=300 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miauZDoze: TXB2=0,2 mg/kg, otopljen u DMSO (10 mg/mL), i.p. 30 min. prije APAP, u V=0.2 mL/miaud anti-TXB2-liofilizirana protutijela otopljena u 5 ml PBS, i.p. 2 sata prije APAP, u V=0,2 mL/miau &W'praenje pre~ivljenja: 2 dana (48 sati)SKUPINE: (N=13) A = F.O. + APAP  B = TXB2 + APAP  =C = anti-TxB2 + APAP   " > A= F.O. + APAP  > ?B= TXB2 + APAP  @C= anti-TXB2 + APAP sati nakon APAP xy)Abr. ~ivih miaeva AAA~ ~ *@~  *@~ *@~ @%~ (@~  *@~ *@ ~  @&~ "@~  &@~ (@ ~ (@'~  @~  $@~ &@ ~ 0@ ~ @ ~  "@~ &@ ~ 4@ ~ @ ~  @~ &@ ~ 8@7z.& & & & & & ..7& & & & & & & & & & & .7{{{~ &@ ~ <@ ~ @ ~  @~ &@ ~ @@| & & 5& & & & & & & .|~  @~ &@ ~ B@~ @~ B@~ &@ ~ D@%~ @~ B@~ &@ ~ F@&~ @~  @ ~  $@  ~ H@3~ %@%~ C@2~ D$@D *@*@*@ "EA-Vehikulum + APAP F B-TXB2 + APAPDC-anti-TXB2 + APAP > broj ~ivih>@>@$@T<Napomena: Ovdje napraviti graf za pre~ivljenje; ovo e biti  @D6is4449j`hhhhh{h}hhhh6 ! " # % & '  > broj mrtvih >$@>$@@ g T/Slika 13A, udru~uje se sa prethodnom slikom 13B 1!postotak pre~ivjelih~ !G7@!Gx $(~?!HQI?!"%";;7@DDd%";;7@DD d%";;S@DD d"## Slika 13A%%Vehicle + APAP%&& TXB2 + APAP&''anti-TXB2 + APAP'1xy.;8T @$(  j  0A@?,K-]`nz^  # $ 2] `|t  <NMM?]   "??3` / (` / )` / *?pп3d23 M NM4 3QQ ;""Q ;%'Q3_ O NM  MM<4E4D $% MP(3O&)Q4$% MP(3O&*Q4FA%l( 3O:F3 b#M43*Y@9@@#M4% [MZ3Oo&(Q ,Survival of mice (%)'43" 444eee   Bh A@"`Gk]`h <*<%ua>@,,,7   dMbP?_*+%M EPSON AL-M2000 Advanced2  4dXXA4 210 x 297 mm XX,4 4 dd @XX*DLLName32=EPOBF7AC.DLLQ`C:\WINDOWS\system32\" dX??U2                          0 0 0 0 0 0       0 0 0 0 0WPokus I-7: Benzylimidazole (BZI) - kem. antagonist TXA2 sintetaze- ALT-"time response" Datum: 30.09.2009 W'Miaevi: C57Black/6, 34 mu~jaka, e" 3 mj. -$Doze: APAP=200 mg/kg i.g. u V=0,5 mL GDoze: BZI= 50,0 mg/kg, otopljen u F.O., 2 h nakon APAP, u V=0.2 mL/miau SKUPINE (N=17):J A = APAP + F.O.  / &B = APAP (200 mg/kg) + BZI (50 mg/kg)   m 2/crveni brojevi su izba eni radi smanjenja STDEV    skupina  oznaka   ALT (U/l)    !A  "A/1  1X@^? ^>J  !A  "A/2  1ȩ@^? ^G GRUPA  ALT STDV SEM, #bili krivi stdev i sem u obje grupe !A  "A/3 1h@^? ^G VA,_E]t@% %BYX@Xx@   !A  "A/4 1`@^? ^G `B,atE]k@%%#Bb c@G@   !A  (A/5 1@^?^G  )A  *A/6 c@^? ^G.T%Napomena: ovdje napraviti graf za ALT   )A  dA/7c@^? ^GeT.Slika15B, udru~uje se sa prethodnom slikom 15A   )A  ,A/8 Oҹ@^?V_YX  )A  dA/9c @^?`ab  )A ,A/10 c$@^? APAP + vehikulum bb  !AeA/11 1,@^?  APAP + BZI   !AeA/12 1d@^?  fB gB/21^@^@  sig 0.001   fB  gB/3 1V@^@  iB  jB/4 ck@^@  iB  kB/5cY@^@  iB  kB/6 c0@^@  iB  kB/7cp@^@`  iB  kB/8 O@^@  iB  kB/9 ct@^@h @D<yO:?QZNLLKrkLhLLKLKL 0! 0" 0# 0$ % & ' ( ) * , - . / 0 1  iB  kB/10  cN@^@ !iB !kB/11 !cv@^@ "iB "kB/12 "cQ@^@ #iB  #kB/13#cr@^@$ % & ' ( ) *^,^-^.^/^0^1^& @7776 F0 (  j  0A@?))]`htX @*   &] `D|t  <NMM?@*]   "??3` / 5` / 8` / :?pп3d23 M NM4 3QQ ;Q ;Q3_ O   MM<4E4 3QQ ; QQ3_ O   MM<4J[x@4 3QQ ; QQ3_ O   MM<4J[x@4D $% MP(3O&8Q4$% MP(3O&:Q4FAgl n 3O2F3 b#M43*@@@#M4% [$MZ3O;&5Q  ALT (U/L)'43" 444eex@x@G@G@e   B A@"`dx]`D  <***<%ua> @7   dMbP?_*+%"??U!   QPokus I-6: Benzylimidazole (BZI) - kem. antagonist TXA2 sintetaze-"pre~ivljenje"Datum: 31.08.2009W'Miaevi: C57Black/6, 24 mu~jaka, e" 3 mj.-$Doze: APAP=375 mg/kg i.g. u V=0,5 mLmDoze: BZI= 50,0 mg/kg, otopljen u F.O., i.p. 30 min. prije (nije prikazano) i 2 h nakon APAP, u V=0.2 mL/miauSKUPINE (N=13):J    A = APAP + F.O.  ( D = APAP + BZI (2 h nakon APAP)  Skup.A=Skup.B&vrijeme proteklo od inj. APAPZ~ >\*@\*@]~ >@\&@\*@]~ > @\$@\(@]~ >(@\@\(@]~ >0@\@\&@]~ >4@\@\&@]~ >8@\@\$@]~ ><@\@\$@]~ >@@\@\$@]~ >B@\@\$@]~ >D@\@\$@eT.Napomena: Ovdje napraviti graf za pre~ivljenje~ >F@\@\$@eT.Slika 15A, udru~uje se sa sljedeom slikom 15B~ >H@\@\$@]]]]APAP + vehikulum%ON.@DDd : 0mC. 1>6<88888888888   APAP + BZI % ;;S@DDd^F@ (  j  0A@?*<P+]`T&htX R<   x +] `@'|t  <NMM?R<] '  "'??3` / 3` / 4` / 6?pп3d23 M NM4 3QQ ; Q ; Q3_ O   MM<4E4D $% MP(3O&4Q4$% MP(3O&6Q4FA%l( n 3O:F3 b#M43*Y@9@@#M4% [cMZ3O|&3Q 0Pre~ivjelih miaeva (%)'43" 444eee   B< A@"`,]`<@( <**<%ua>@///7   dMbP?_*+%M EPSON AL-M2000 Advanced2  4dXXA4 210 x 297 mm XX,4 4 dd @XX*DLLName32=EPOBF7AC.DLLQ`C:\WINDOWS\system32\" dXX??U}  =                           rPokus I-5: anti-TXB2 protutijela - odreivanje "ex vivo" produkcije 11-dehidro-TXB2 u homogenatima jetre i plazmi19U Datum: 23.09.2009 S%Miaevi: CBAT6/T6, 24 mu~jaka, e" 3 mj. -$Doze: APAP=160 mg/kg i.g. u V=0,5 mL ODoze: anti-TXB2 (40,0 g/kg), otopljen u F.O., 2 h prije APAP, u V=0.2 mL/miau SKUPINE (N=6):J /&A = F.O. + APAP (homogenati jetre)  1 (B = anti-TXB2 + APAP (homogenati jetre)  VV tVehicle + APAP# JC = F.O. + APAP (plazma)  WW tAnti-TXB2 + APAP& JD = anti-TXB2 + APAP (plazma) WW Vehicle + APAP WWW anti-TXB2 + APAP  tNormal XYSkup. AYSkup.BJSkup.CJSkup.D W .Z%koncentracija 11-dehidro-TXB2 (pg/mL)ZZ [r@[R@\@j@\b@  Plasma [S@[f@\@j@\@T@  Liver fragments [e@[N@\ l@\@j@  [^@[R@\n@\R@  [X@[e@\b@\[@  [s@[f@\d@\@T@  i0average x razrjeenje (x6 za A i B; x3 za C i D)#f@ %B#^@ %B#i@ %B#]@ %Br@r@  STDEV#Kz:Y@ %B #'&M@ %B #0"A@ %B #p J@ %B rr  SEM*E@@T@@r@r@  T|Napomena: ovdje napraviti graf (usporeuju se skupine A i B te C i D); ato se ti e naziva osi napravi kako ti je najzgodnije TSlika 17 [r@? 8v :sM7Oj^_9/mXS\BBBB7V+; ! " # $ % & ' ( ) * + , - . / 0 1 2 3 4 5 6 <  [S@? ![e@? !"[^@? "#[X@? #$[s@? $%[R@@ %&[f@@ &'[N@@ '([R@@ ()[e@@ )*[f@@ *+\@j@@ ++Komentar: Samo dodati po 1 stupac lijevo koji bi se odnosio na normalne miaeve u podioke plazma i homogenati. Naime, konc. TXB2 u plazmi i homogenatu normalnih miaeva je 1-2 pg/mL. Ne trebaa stavljati nikakve zvjezdice niti ata drugo, nego samo dodati po ,\@j@@ ,-\ l@@ -.\n@@ ./\b@@ /0\d@@0 1\b@@1 2\@T@@2 3\@j@@3 4\R@@4 5\[@@5 6\@T@@6 <s 4&&&&&&&&&&&/&&&&6666666P 0 (  j  0A@? $ $]`P.n^ .  # / =-] `'@6667   dMbP?_*+%"??U$                              LPokus: I-9 (Daltroban-kemijski antagonist TXA2r) - odreivanje pre~ivljenja Datum: 06.06.2009 U&Miaevi: CBA/ T6T6, 26 mu~jaka, e" 3 mj. MDoze: APAP=300 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miau Doze: Daltroban 5,0 mg/kg, otopljen u TRISu (5 mg/3mL), potom razrijeen u PBS-u i dat i.p. 30 min. prije APAP, u V=0.2 mL/miau SKUPINE (N=13): A = F.O. + APAP 2 =)B = Daltroban (30 min. prije APAP) + APAP =DDD  =DDDD  K  J Skup. A Skup. B  broj ~ivih >@p"@   ! pre~ivljenjeSkup.A=Skup.B  broj mrtvih >@3@  &vrijeme proteklo od inj. APAPZ1postotak pre~ivjelih GK7? Ge`TR'?   ~ >\*@\*@! ;;G@  d! NNQ@   d  > ~ >@\(@\*@G ~ > @\$@\(@ TENapomena: Nije potrebno ra unati, jer nema statisti ki zna .razlike;   ~ >(@\"@\(@ QT$Ovdje napraviti graf za pre~ivljenje  ~ >0@\ @\&@ gT/Slika 16A, udru~uje se sa prethodnom slikom 16B  ~ >4@\@\&@] ~ >8@\@\"@] ~ ><@\@\"@] ~ >@@\@\"@ ]Vehicle + APAP ~ >B@\@\"@ ]Daltroban + APAP ~ >D@\@\"@] ~ >F@\@\"@] ~ >H@\@\"@] ]] ] >H0>y+<<V""NcRRRRuwRRR$" #  "] #]]Dp$(  $p $ 6NMM?` .] ;  M EPSON AL-M2000 Advanced2  4dXXA4 210 x 297 mm XX,4 4 dd @XX*DLLName32=EPOBF7AC.DLLQ`C:\WINDOWS\system32\" dX??3` / 9` / =` / >?pп3d23 M NM4 3QQ ;  Q ; Q3_ O   MM<4E4D$% MP(3O&=Q4$% MP(3O&>Q4FA5b n 3O^9G3 b#M43*4@#M4% MZ3Oo&9Q ,Survival of mice (%)'43" 444eee >@5557   dMbP?_*+%M EPSON AL-M2000 Advanced2  4dXXA4 210 x 297 mm XX,4 4 dd @XX*DLLName32=EPOBF7AC.DLLQ`C:\WINDOWS\system32\" dX??U                              0 0           0 0 \Pokus: I-8 (Daltroban-kemijski antagonist TXA2r) - odreivanje ALT-a (20-22 h nakon APAP-a)Datum: 03.06.2009U&Miaevi: CBA/ T6T6, 16 mu~jaka, e" 3 mj.MDoze: APAP=150 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miauDoze: Daltroban 5,0 mg/kg, otopljen u TRISu (5 mg/3mL), potom razrijeen u PBS-u i dat i.p. 30 min. prije APAP, u V=0.2 mL/miauSKUPINE (N=8):A = F.O. + APAP2 =)B = Daltroban (30 min. prije APAP) + APAP =DDD =DDDD K _ 2(crveni brojevi su izba eni iz statistike   skupina  oznaka   ALT (U/l)  Stupanj hemolize  ;Average (kad se izbaci najvea vrijednost iz svake skupine)   !A "A,/1 ~ L$@#~ ?grupa ALTSTDEV SEM !A "A,/2 ~ Lֺ@#~ ? %A(۶m۶S@%$B([Ĥ0ޱ@%$B ~  A !A "A,/3 ~ L@$~ ? &B,m۶mi@%%B,~x˕@%%B ~  A !A "A,/4 ~ Lº@#~ ?  !A (A,/5 ~ L@#~ ?J !A (A,/6 ~ L,@#~ ?.T%Komentar: Ovdje napraviti graf za ALT  )A *A,/7 ~ O4@O~ ?eT.Slika 16B, udru~uje se sa sljedeom slikom 16A  )A ,A,/8 ~ M&@+~ ? 7B  8B/1 ~ np@n~ @ 7B 9B,/2 ~ n@n~ @Vehicle + APAP  7B 9B,/3 ~ n @n~ @Daltroban + APAP  7B 9B,/4 ~ n>@n~ @ 7B 9B,/5 ~ NC@N~ @ NS p=0.07  :B ;B,/6 ~ o@o~ @ :B <B,/7 ~ o@o~ @ :B <B,/8 ~ o`@o~ @>08s%56PYYYXz|YuYYY (   p  6NMM?p( +] 8  "8??3` / 7` / ;` / <?pп3d23 M NM4 3QQ ;Q ;Q3_ O   MM<4E4 3QQ ;QQ3_ O   MM<4J[fffff@4 3QQ ;QQ3_ O   MM<4J[fffff@4D $% MP(3O&;Q4$% MP(3O&<Q4FAl n 3O2F3 b#M43*p@#M4% [$MZ3O;&7Q  ALT (U/L)'43" 444eefffff@fffff@@@e >@---7   dMbP?_*+%"??U6                              0 0           0 0 0  WPokus: I-4 (U-46619-stabilni agonist TXA2r) - odreivanje ALT-a (20-22 h nakon APAP-a) Datum: 08.03.2008 Q$Miaevi: CBA/ T6T6, 32 ~enki, e" 3 mj. MDoze: APAP=150 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miau 9Doze: U-46619 0,2 mg/kg i 0,8 mg/kg, isparen pod nje~nom strujom N2, otopljen u PBS-u (2 mg/mL), i.v. 30 min. prije i 2 sata nakon APAP, u V=0.2 mL/miau SKUPINE (N=8): A = F.O. + APAP : 1B = U-46619 0,2 mg/kg (30 min. prije APAP) + APAP 22 : 1C = U-46619 0,8 mg/kg (30 min. prije APAP) + APAP 222  D = U-46619 (sam) K    Vehicle + APAP skupina  oznaka   ALT (U/l)  Stupanj hemolize  }srednja vrijednost w  # U-46619 (0.2 mg/kg) + APAP !A "A,/1 ~ L@#~ ?grupa ALTSTDEV SEM # U-46619 (0.8 mg/kg) + APAP !A "A,/2 ~ L@#~ ?% Vehikulum#@"@ %B#Q 5!@ %B ~  QO A    !A "A,/3 ~ LT@$~ ?*&! U-46619 (0.2 mg/kg)#0#@ %B#}|ak@ %B ~  A !A "A,/4 ~ L@#~ ?*&! U-46619 (0.8 mg/kg)#@ %%B#|ǂ[&@ %%B ~  @ !A (A,/5 ~ L@#~ ?o'3D-ovo ne treba ii na graf, spomenut u to u tekstu#*H@ %&+B#4$@ %&+B ~  `z@ !A (A,/6 ~ L@n@#~ ?  )A *A,/7 ~ M@+~ ?J )A ,A,/8 ~ M4@+~ ?TiKomentar: Ovdje je o ito da U-46619 nema nikakvog u inka, pa ne treba niata ra unati- samo napraviti graf  .B  /B/1 ~ N@#~ @gT/Slika 14B, udru~uje se sa prethodnom slikom 14A  .B 0B,/2 ~ N@$~ @ .B 0B,/3 ~ N@1~ @ .B 0B,/4 ~ N[@#~ @ .B 0B,/5 ~ N4@#~ @ 4B 5B,/6 ~ O@+~ @ 4B 6B,/7 ~ Ov@+~ @ 4B 6B,/8 ~ OJ@+~ @ .C /C,/1 ~ N@N~ @ .C 0C,/2 ~ N@N~ @@nD<s[9:\\<9YY6YYYYYYYY  !  "  #  $  %  &  ' 0( 0) 0* 0+ 0, . / 0 1 2 3 4 5  .C  0C/3 ~ N@ N~ @ !.C  !0C/4 ~ !N@!N~ !@ ".C  "0C/5 ~ "N @"N~ "@ #.C  #0C/6 ~ #N@#N~ #@ $.C  $0C/7 ~ $N0@$N~ $@ %.C  %0C/8 ~ %NE@%N~ %@ &:D &;D/1 ~ &PI@ &+ ':D 'QD/2~ 'PN@ '+ (:D (QD/3~ (PF@ (+ ):D )QD/4~ )PK@ )+ *:D *QD/5~ *PI@ *+ +:D +QD/6~ +P>@ ++,.RRSS/RRSS0RRSS1RRSS2RRSS3RRSS4RRSS5RRSS.MFFFFFF;:::::(  p  6NMM?@!9 /] D>  "D>??3` / +` / .` / 0?p@3d23 M NM4 3QQ ;Q ;  Q3_ O   MM<4E4 3QQ ;QQ3_ O   MM<4J[= ףpz@4 3QQ ;QQ3_ O   MM<4J[= ףpz@4D$% MP(3O&.Q4$% MP(3O&0Q4FA 3Og69j3 b#M43*@p@#M4% MZ3O;&+Q  ALT (U/L)'43" 444ee= ףpz@= ףpz@{Ga@{Ga@@@e > @555  7   dMbP?_*+%"??U} I "     \Pokus: I-3 (U-46619-stabilni agonist TXA2r) - odreivanje pre~ivljenja miaeva-dose response&Datum: 08.03.2008&Q$Miaevi: CBA/ T6T6, 32 ~enki, e" 3 mj.&MDoze: APAP=250 mg/kg i.g. u V=0,5 mL; F.O. 30 min. prije APAP u V=0,2 mL/miau&9Doze: U-46619 0,2 mg/kg i 0,8 mg/kg, isparen pod nje~nom strujom N2, otopljen u PBS-u (2 mg/mL), i.v. 30 min. prije i 2 sata nakon APAP, u V=0.2 mL/miau&]*Pre~ivljenje: kroz 48 h od primjene APAP-a&SKUPINE (N=12):Vehicle + APAP A = F.O. + APAP#U-46619 (0.2 mg/kg) + APAP : 1B = U-46619 0,2 mg/kg (30 min. prije APAP) + APAP # U-46619 (0.8 mg/kg) + APAP : 1C =U-46619 0,8 mg/kg (30 min. prije APAP) + APAP& 2222q I  (@(@(@  ( I I Skup. A Skup. B Skup.C 2  V Vehikulum 2U-46619 (0.2 mg/kg) 2U-46619 (0.8 mg/kg)  =broj pre~ivjelih ~ivotinja broj ~ivih  >@> @>@  'vrijeme nakon inj. APAP (sati)>>>> broj mrtvih  >@>@>@  ~ >>>(@>(@>(@ 1postotak pre~ivjelih  G7d? GioT? G7d? >>>>~ >@GG jedanaestG jedanaestG jedanaest % *M@D D d% P@D D d% *M@D D d >>>>~ > @>Gdevet> jedanaest> jedanaestGGGG~ >(@>G sedam>deset> jedanaest~ >0@>G sedam>deset>deset~ >4@>G sedam>deset>desetlm Komentar: llllllllll~ >8@>G sedam >osam>deset llQm$Slika 14A, udru~uje se sa slikom 14B lllllllll~ ><@>Gsedam >osam >osam~ >@@>G sedam >osam >osam~ >B@>G sedam >osam >osam~ >D@>G sedam >osam >osam~ >F@>G sedam >osam>sedam~ >H@>G sedam >osam>sedam >@> @>@((>B XHgiuhL,xuqnoooppL, ! >!G<  (   p  6NMM?p ,Z] A  "A??3` / ,` / -` $5C?p@3d23 M NM4 3QQ ; Q ; Q3_ O   MM<4E4D$% MP(3O&,Q4$% MP(3O&-Q4FA  3O < 3 b#M43*T@9@?#M4% 3MZ3Oo&CQ ,Survival of mice (%)'43" 444eee > @0 00 7 -  --'-- --'-- --'-- N--'-- N-- $bbb$PPPbVVVwwt{bnnnwwt{--'-- N--'-- -N NArial-JNJNvJvNWJWN9J9NJNNNN--"System<96-'--- ---'---    2 =0 2 (1000 2 n(2000 2 O(3000 2 1(4000 2 (5000---'--- ---'---  2 APAP + vehikulum 2 Z APAP + BZI---'---  Arial--------'--- &K  Arial-2  ALT (U/L)I ------'--- ---'--- ---' ' 'NANIOh+'0@HXh UserUserMicrosoft Excel@WF,u@mIgr՜.+,0@ PDocumentSummaryInformation87p1TableMSummaryInformation(=PDocumentSummaryInformation8`=Xh px HOME  Sheet1Sheet2Sheet7Sheet6Sheet5Sheet9Sheet8Sheet4Sheet3Chart4  Worksheets ChartsOh+'0 P\lx     bs EmH݊l[JEBQO3P[!zuJŋ46sEQ9Tn'{Ʈce/+u'gSc(c2wZ澑;Slzd'4'_WW6;ɩ3L}(m(s`z99Ei5%!LƘLQX.$(P>uĞ QOm9>Xq1m4igl߯YȮ;m$soVڧ|?%~På/*BOh*3?rk1:oS2,, +IUG{-QqMn.mux>o[d~?ڌ?gv!x_z~9ތa09E:Q/[jӹq0cNXd&15l'/n:4 :SIa7<};ogg’͛~jn7ǚE|y1Cd^\5kOijE÷hxi1' [-㫵7;K_qY=|ir7"O< ʗŜscĨWy.q>8x^/NYCj+:w ?JC |S?u%})T+ cp㎡6Jưwk9ݗ_]x_>DmZORL/D>/ޤ}-6|B>Tӄ x-E<]MwL?0~ ӭЉM/7/bMh3BvsքHOd7[M(KA q7[V aрf,`Tm-7Lkz^gAWrcۑyb-cz; Llz~_޻|o1WffGqaJcϸwsI'Overdoses of the analgesic and antipyretic acetaminophen represent one of the most common pharmaceutical product poisonings in the United States today (Litovitz et alUserNormalFilip136Microsoft Office Word@@1@X@. 30  F'Microsoft Office Word 97-2003 Donih.gov/pubmed?term=%22Negita%20M%22%5BAuthor%5Dv:Hhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Kobayashi%20T%22%5BAuthor%5Dm3Ghttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yokoyama%20I%22%5BAuthor%5D#9,http://www.ncbi.nlm.nih.gov/pubmed/12928598M05javascript:AL_get(this, 'jour', 'Transplantation.');d<Hhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sugimachi%20K%22%5BAuthor%5D QFhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Shirabe%20K%22%5BAuthor%5Dl>Hhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Kishikawa%20K%22%5BAuthor%5DDFhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Itasaka%20H%22%5BAuthor%5DEEhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yanaga%20K%22%5BAuthor%5D@Fhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Suehiro%20T%22%5BAuthor%5D<~0javascript:AL_get(this, 'jour', 'J Surg Res.');d<{Hhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sugimachi%20K%22%5BAuthor%5DH xEhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Payne%20WD%22%5BAuthor%5Dq3uChttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Chen%20S%22%5BAuthor%5Dg&rHhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Shinagawa%20Y%22%5BAuthor%5DRoBhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Kin%20S%22%5BAuthor%5D QlFhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Shirabe%20K%22%5BAuthor%5D!1i,http://www.ncbi.nlm.nih.gov/pubmed/20120521'3f,http://www.ncbi.nlm.nih.gov/pubmed/20503171wNc4javascript:AL_get(this, 'jour', 'Prostaglandins.');i7`Ghttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Kasahara%20M%22%5BAuthor%5Dk:]Chttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Koda%20A%22%5BAuthor%5Do?Z՜.+,D՜.+,T hp  HOMER_ Overdoses of the analgesic and antipyretic acetaminophen represent one of the most common pharmaceutical product poisonings in the United States today (Litovitz et alOverdoses of the analgesic and antipyretic acetaminophen represent one of the most common pharmaceutical product poisonings in the United States today (Litovitz et al TitleNaslov: 8@ _PID_HLINKSA:9 /javascript:AL_get(this, 'jour', 'Fiziol Zh.');?mKhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Aleksieieva%20IM%22%5BAuthor%5D4|Ghttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Makohon%20NV%22%5BAuthor%5D)+Ohttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Korni%C4%ADchuk%20HM%22%5BAuthor%5D!hGhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hrushka%20NH%22%5BAuthor%5DS~Njavascript:AL_get(this, 'jour', 'Prostaglandins Leukot Essent Fatty 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