Effects of 1-morpholinoacetyl-2-methyl-3-phenyl-4-oxo-1, 2, 3, 4-tetrahydroquinazoline hydrochloride (HQ-275) on acute experimental hepatic injury induced by carbon tetrachloride in rats.

Pharmacological effects of HQ-275 on CCl4-liver injury ha been in vestigated, and the results compared with those of rats treated with CCl4 plus HQ 275 with those of normal rats and only CCl4-treated rats. In the excretion test of biliary flow, bilirubin, solid contents and B&bull;S&bull;P, rats treated with CCl4, alone or with CCl4, plus HQ-275 showed less excretion than those of normal rats, and the differences among these three categories were not significant. HQ-275 revealed a potent recovery process from the liver damage caused by CCl4, in both histological and biological levels. Changes of Na+ and K+ in bile, water content of liver and body temp. were recognized among the three groups. It can be concluded from over-all results that there may be some differences between structural and functional recovery processes from CCl4-hepatotoxicity. In a previous report from our laboratory, 1-morpholinoacetyl-2-methyl-3-phenyl-4 oxo-1, 2, 3, 4-tetrahydroquinazoline hydrochloride (HQ-275) has been demonstrated to have a potent inhibitory effect against experimental hepatic injury in rats by carbon tetra chloride (CC14) with respect to histological findings (1). CCI, is the agent most widely used to produce experimental liver damage in laboratory animals. Due to the importance of CC14 hepatotoxicity as a model for liver disease and damage, it is of interest to investigate the effect of HQ-275 on this toxicity regarding hepatic transport processes that influence biliary excretion. This paper, covers a more detailed investigation of HQ-275, choleretic actions, phenol tetrabromophthaleinsulfo nate (B . S . P)-excretion and some liver function using CC14-intoxicated rats. In addition, influence of HQ-275 on the developmental or recovery processes of hepatic injury, (administered to rats before or after CCI4-poisoning) were also studied employing transaminase levels (s-GOT and s-GPT) and histological findings as indices to evaluate the degree of the liver lesions. MATERIALS AND METHODS Male rats of Wistar strain (200-250 g) were mainly used for all experiments. In the series, six animals were employed as one group. They were fed on a commercial diet (CLEA, CA-1) and water ad libitum. CC],, suspended as a 10% solution in olive oil, was administered to rats orally in a dose of I g/kg (0.64 ml/100 g in olive oil) for the pur pose of creating severe hepatic injury (2). HQ-275 was dissolved in physiological saline. Six non-treated (Gr.: A), served as control, and 18 HQ-275-treated animals were challenged with CC1,. The latter category was divided into three groups (Gr.: B HQ-275 10 mg/kg, p.o., Gr.: C 30 mg/kg, p.o. and Gr.: D 60 mg/kg, p.o., respectively). All doses of HQ-275 were administered one hr before and one hr after CCI, as a single oral admin istration for 2 days, respectively. In addition, six normal rats (Gr.: E) were prepared as control along with the other four groups. During 6-8 hr after the second CCI,-poison ing, the animals were all sacrificed except for those utilized for investigation of the influ ence of HQ-275 on histological developmental and recovery processes of CC], liver injury. Biliary excretion: For the biliary excretion tests, the common bile ducts of the rats, anesthetized throughout with urethane (1.2 g/kg, s.c.), were cannulated centrally so that the bile could be collected via a polyethylene tube. The cystic duct was ligated and the midline abdominal incision was sutured. The animals were maintained in a relative hu midity of 60% at a room temp. of 24-: l °C. After the biliary outflow had reached a steady state, the drugs were administered into the femoral vein. Measurement of the biliary flow was made every 30 min after administration of the drugs (3, 4). Bilirubin and solid contents: Bilirubin and solid contents which were contained in the biliary volume excreted were assayed by the modified Evelyn-Malloy's method and by the method of dry wt., respectively (5). The outflow of the bile through common bile ducts was collected in a graduated pipette. After measurements, half of the collec tions every 30 min were used to measure bilirubin concentration and the remaining bile was dried for 10 hr in a constant oven at 105-110 'C after which each dry residue was weighed and concentration determined. B • S • P-excretion: For examination of the coloring matter excretion, B • S • P was in jected into the femoral vein by a single injection in a dose of 20 mg/kg over a 30-sec period and at 3, 5, 10 and 15 min after the administration of B • S • P, the concentration of B • S • P in serum was measured according to the method of Rosenthal and expressed as a percent age (6). Biochemical assays: Serum samples for the estimation of transaminase levels (GOT and GPT) were obtained from blood collected from rats by excising V. Jugularis under ether anesthesia just before sacrifice. The levels of transaminases were measured using the method of Reitman and Frankel and expressed in Karmen units (7). Histological findings: It is well known that there is no direct relation between two degrees of liver lesions and the serum enzyme levels, therefore histological changes in the liver were also examined with a light microscope. A portion of liver from individual ani mals was fixed in 10% phosphate-buffer formalin and acetone and stained with sudan III, hematoxylin and eosin for microscopical studies. The extent of the necrosis produced by CCI,, (focal areas of centrilobular necrosis and ballooning) was measured using an Amsler type planimeter on a microscopical photograph of low magnification. For histochemical analysis, alkaliphosphatase and acid phosphatase activity in the liver section was measured according to the method of Gomoris (8). Furthermore, ap pearance of glycogen in liver tissue was demonstrated by the method of PAS-reaction (9). Relationship between administration time of HQ-275 and histological and biochemical activity: For the purpose of examining whether or not HQ-275 exerts a therapeutic ac tion on the damaged liver and/or the preventing effect, the drug was given to rats at various time intervals before and after CCI, administration. In this experiment, both histological changes of the liver such as fatty and hydrotropic alterations of the cells and the changes of transaminase levels were examined. Rats were administered p.o. 30 mg/kg of HQ 275, at 1, 3 and 6 hr prior to, or 1, 3, 6 and 12 hr after an oral administration of 1 g/ kg of CC],. Blood was collected 24 hr after CC1,-poisoning. The serum transaminase levels were measured at various time intervals, respectively. Histological and biological assays were carried out according to the method mentioned above. It is well recognized that the effect of CC], on the liver is modified by numerous factors either in the direction of aggravation or alleviation (10). The authors therefore examined the following actions among the groups of normal (Gr: E), only CCI, treated (Gr.: A) and CCI, plus HQ-275 treated animals (Gr.: C). FIG. 1. Dose-response curves of HQ-275 in normal and CC1,-treated groups of rats. Basal biliary flow of each group taken as 100%, respectively. Biliary flow, collected for 0-30 min after administration of the drugs, was measured. F] : normal (Gr.: E), O : CCI, plus HQ-275 (10 mg/kg, p.o., Gr.: B), A : CCI, plus HQ-275 (30 mg/kg, p.o., Gr : C) and N : control (CCI, I g/kg, p.o., Gr. A). Each vertical line indicates the S.E. of six animals. *, indicates the values to be significantly different from controls. (P<0.05) Chemical analysis of Na+ and K+ in bile: Na+ and K+ in bile were determined by means of a Hiranuma flame photometer. Estimation of the biliary Na' and K' con centrations was calculated from the Na+ and K' content in bile. Water content of liver: The water content of liver was determined by drying the tissue for 10 hr in a constant oven at 105-110'C to constant wt. Body temp.: It has recently been shown that one alteration in biliary excretion is the fact that it is temp. dependent (11). Statistical analysis: A statistical comparison of the data was performed employing the Student's t test. Values of P<0.05 were considered to be representative of significant differences between means. RESULTS Effects on biliary excretion FIG. 2. Upper curves (solid lines) : choleretic activity as % increase of biliary flow. Middle curves (dotted lines) : bilirubin concentration. Lower curves (solid lines) : solid content concentration. L] : normal (Gr. : E), O : CC14 plus HQ 275 (10 mg/kg, p.o., Gr. : B), L : CC14 plus HQ-275 (30 mg/kg, p.o., Gr : C) and • : control (CC14 1 g/kg, p.o., Gr. : A). Maximum S.E., upper curves : 14.2%, middle curves : 11.6% and lower curves : 10.8%. Each point represents the mean of six rats. Other conditions as in Fig. 1. The results are represented in Figs. 1 and 2. It is evident that in all doses of HQ-275, biliary flow was maximum during the first 30 min. As shown in these figures, rats treated with CCI, alone (Gr.: A) showed a marked decrease in biliary volume, bilirubin and solid contents excretion when HQ-275 was administered i.v. in any dosage. As shown in Fig. 2, the rats treated with CCI, plus HQ-275 (Gr.: B) did not show an increase in biliary flow such as was seen in normal rats. When 10 nag kt, of HQ-275 was injected i.v. to rats pre treated with CCI, Plus HQ-275 (Gr.: C) and to the rats treated with CCI, alone (Gr.: A) comparable effects were seen regarding excretions of bile, bilirubin and solid contents. B • S • P-cxcrct ion I n the dye excretion test, concentrations of B • S • P remained in the blood after admin istration of B • S • P (20 mg k(y, i.v.) in a single injection as seen in Fig. 3. There were sig nificant changes in the average concentration of B • S • P in the serum at 3-15 min after administration as compared to the normal rats (Gr.: E) and only the CCI,-intoxicated rats (Gr.: A). Regarding time-courses of B•S•P-disappearances in the serum, the rate of only the CCI,-intoxicated rats was approx. 2-2.5 times later than that of normal rats. In rats treated with CCI, plus HQ-275 (Gr.: B-D, respectively) the rate was comparable to that seen in rats treated with CCI, alone (Gr.: A). Fiu. 3. Biliary excretion of B•S•P in rats. Single injection of B•S•P was admini stered at I min. : normal (Gr.: E) , : CC], plus HQ-275 (10 mg `kg, p.o., Gr. : B), : CCI; plus HQ-275 (30 nmg~kg, p.o., Gr.: C) and • : control CCI, 1 g kg, p.o., Gr.: A). Each point represents the mean S.E. of six rats. Other conditions as in Fig. 1. E(f ccc'ts of HQ 275 a,,uiirst CCl,-into.vicution on the liver dama,,c (a) Histolo,,ica/ /inding.~ : The results presented in Fig. 4 are in accord with the histological evaluation of the livers. Rats treated with CCI, Plus HQ-275 (Gr.: C) did not modify to any extent the normal structural pattern of the liver (I: a-c). Rats treated with CCI, alone (Gr.: A), however, produced an intense centrilobular necrosis associated FI(G. 4. (l-a--c) : Liver section taken from a rat treated with CCI4 plus HQ-275 30 mg,kg, p.o. Centrilobular necrosis and ballooning degeneration is limited to small areas (1-a). Glycogen granules are seen in the central zone (l-b). Acid phosphatase activity was observed and compared with that of control (I-c) . (11-a c) : Liver section of a rat which had been given CC]., alone. Note the striking centrilobular necrotic and ballooning (11-a), glycogen granule deposition (II-b) and decrease of acid phosphatase activity (1T-c). a : Hemato xylin and eosin, x 100. b : PAS, x 100. c : Gomori's reaction, a 100. with hemorrhage and edema; groups of cells bordering the necrotic areas presented bal looning. The liver cells in the periportal zones were enlarged and in that area the normal structural pattern was lost (11: a). Acid or alkali phosphatase activity and the changes of glycogen granule in the liver were also examined. In rats treated with CCI, alone (Gr.: A), there was a slight glycogen granule deposition in the liver cells of the periportal zones 01: b), however, there was a fairly good number of glycogen granules remaining in rats treated with CCI, Plus HQ-275 (Gr.: C) (1: h). Furthermore, acid phosphatase activity decreased in rats treated with CCI, alone (Gr.: C) (I1: c). On the contrary, rats treated with CCI, plus HQ-275 (Gr.: C) showed a marked protective effect against the decrease of acid phosphatase activity in only the CCI, intoxicated animals (Gr.: A) (I: c). On the other hand, an effect of HQ-275 on alkali phosphatase activity was not observed. (h) Biochemical activiti : As shown in Fig. 5, administration of CCI, alone re sulted in a marked increase in s-GOT and s-GPT levels. This increase was partially pre vented by administration of HQ-275 30 mgikg, p.o. Furthermore, rats treated with CC1, plus HQ-275 (Gr.: D) revealed a similar activity to that of normal rats. It is very sig nificant for clarification of the action mechanism of HQ-275 that the biological effect of this compound was evidenced with oral administration, and that elevation in serum trans aminase levels after CCI,-poisoning was markedly prevented by an oral dosage of 30 mg/ kg of HQ-275 (Gr.: C), and also partially prevented even with a small oral dosage of 10