Protective effect of Ginkgo biloba extract on liver damage by a single dose of CCl(4) in male rats.

Functional and morphological alterations were generated by p.o. (per os) administration of a single oral dose of carbon tetrachloride (CCl(4); 0.125 mL/kg b.w., equivalent to 293 mg/kg) to adult male Wistar rats. CCl(4) significantly increased (p < 0.05) the serum activities of alanine aminotransferase (ALT; 7478 ± 1044%) and aspartate aminotransferase (AST; 6964 ± 833%), compared to control rats; CCl(4) also significantly decreased serum concentration of albumin (23 ± 5.5%) and increased the concentration of malondialhdeyde (MDA) in liver (300 ± 33%). Furthermore, CCl(4) down-regulated the mRNA steady-state level of tumor necrosis factor a(TNF-a). CCl(4) produced necrosis in the central lobe area, extended to the periphery, nuclear alterations (pycnosis, karyolysis and karyorrhexis), and cytoplasmic acidophilia. The pretreatment with 4 mg/kg (p.o.) of Ginkgo biloba extract (GbE), for 5 days, prevented most of the damage caused by CCl(4): significantly decreased the serum activities of ALT and AST (54 and 65%, respectively), compared to CCl(4)-treated rats; GbE partially prevented the increase of liver MDA (55 ± 14%) and the decrease of albumin concentration to 12 ± 0.2%. This pretreatment prevented the down-regulation of TNF-a and up-regulated the interleukine 6 (IL-6) mRNA steady-state level. Moreover, the GbE reduced the amount of necrotic areas in the central lobe area, compared to CCl(4)-treated rats.

Autofluorescence as a tool to study mucus secretion in Eisenia foetida.

Autofluorescence in living cells is due to the presence of endogenous substances that emit fluorescence upon excitation by incidental light. A type of fluorescence, bioluminescence, has been suggested to be linked to mucus secretion in earthworms; however, the origin and the physiological function of this fluorescence are not clear. The aims of this work were to describe autofluorescence in the earthworm Eisenia foetida by SEM, CLSM, and fluorescence microscopy and to examine the possible mechanism of mucus secretion by video microscopy. Earthworms were stimulated either chemically or electrically to induce the secretion of yellow mucus, which was subsequently studied by video microscopy. Mucus was released from the body wall and near the mouth. This phenomenon was associated with autofluorescence and involved at least four distinct stages: release of vesicles, formation of granules, muscular contraction, and organization of strands. The fluorescent molecules were stored in vesicles bound to the membranes. These vesicles were intact when shed from the body. The vesicles were stable but also changed to a granular material or formed strands. Video analyses demonstrated that secretion was dependent on the type of stimulus.

The ATP levels in kidneys and blood are mainly decreased by acute ingestion of tullidora (Karwinskia humboldtiana).

Our previous acute toxicity studies with Karwinskia humboldtiana (Kh) in rats showed renal hemodynamic changes with a marked increase in the fractional excretion of sodium and morphological damage. To analyse the effects of Kh or 'tullidora' on energetic metabolism, a single dose of an oral preparation from the seed fruits was given to Wistar rats (1.25 g/kg). In tullidora-treated rats there was 8% mortality. ATP concentrations in renal tissue decreased significantly (control: 53.85+/-3.34, tullidora 38.28+/-5.31 micromol/g fresh tissue, P<0.05). Total blood (54.8+/-0.96, tullidora: 40.2+/-1.55 micromol/dL, P<0.01) and haemoglobin-ATP concentrations (3.69+/-0.12, tullidora: 2.56+/-0.11 micromol/g, P<0.01) were also significantly diminished. Moreover, the total protein in renal cortex from tullidora-treated rats decreased as compared to control group (control: 71.43+/-2.88, tullidora: 55.20+/-4.06 mg/g fresh tissue, P<0.05). In contrast, Na+-K+-ATPase activity in tullidora-treated animals was not different from control rats. These findings might partially explain the acute effects and mortality observed in the Kh treated rats.

Glutathione S-transferases and esterases in placenta after normal and pre-eclamptic pregnancies.

Severe pre-eclampsia reduced significantly (P<0.05) by 68+/-6 per cent (mean+/-sem, n=10) the maximal velocity (V(max)) and, consequently, reduced significantly by 60+/-7 per cent the catalytic efficiency (C(E)) of placental glutathione transferase pi, assayed with ethacrynic acid. Mild and severe pre-eclampsia reduced significantly by 82+/-5 per cent (mean+/-sem, n=5) and by 41+/-5 per cent (mean+/-sem, n=10), respectively, the V(max)and, consequently, reduced significantly by 72+/-7 and by 33+/-13 per cent, respectively, the C(E)of esterase, assayed with p-nitrophenyl acetate. Furthermore, severe pre-eclampsia increased significantly by 296+/-78 per cent the Michaelis-Menten constant (K(m)) of total GST, assayed with chlorodinitrobenzene and, consequently, decreased significantly the C(E)by 83+/-3 per cent. On the other hand, the concentrations of total and non-protein thiols did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. These findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women suffering pre-eclampsia, mainly in the severe phase.

Immediate and delayed effects of lead on AChE, GSH-T and thiols in the substantia nigra, neostriatum and cortex of the rat brain.

We studied the effects, at 10 and 30 min, of a single dose (10 mg kg(-1)) of lead chloride, administered by the intraperitoneal route, on the activities of acetylcholinesterase (AChE) and glutathione transferase (GSH-T) and on the concentrations of total and non-protein thiols in substantia nigra compacta (SNCO) and substantia nigra reticulata (SNRE), caudate putamen (CAU) and cerebral cortex (CC) from adult male rats in comparison with the effects of this metal at 24 and 72 h. The main immediate effects of lead consisted of decreased GSH-T activity and total and non-protein thiol concentrations in CAU and CC 10 min after administration. These effects were reversed after 30 min but with increased GSH-T activity in SNCO and AChE activity in SNRE along with diminished concentration of homogenate proteins in SNRE, CAU and CC. The GSH-T activity again was increased in SNCO but the AChE activity was decreased in CC 24 h after Pb administration; total and non-protein thiol concentrations were diminished but homogenate protein concentration was augmented in all areas. Finally, 72 h after Pb administration, AChE and GSH-T activities were decreased in CAU and CC, accompanied by an increased concentration of precipitate and supernatant proteins; supernatant protein concentration also was augmented in SNCO and SNRE; here, again, the concentrations of total and non-protein thiols were diminished and the homogenate protein concentration was augmented in all areas.

The activities of six exo-and endopeptidases in the substantia nigra, neostriatum, and cortex of the rat brain.

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and gamma-Glutamyl transpeptidase (gammaGTP), and two endopeptidases: Postproline endopeptidase (PEP) and "Trypsin-like" peptidase ("T-L" P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) gammaGTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a "Trypsin-like" peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.

Sex differences in the enzymatic hydrolysis of acetylsalicylic acid by microsomes from various rat tissues.

We studied the in vitro hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) catalysed by microsomal preparations from liver, kidney, small intestine and stomach mucosas and blood serum of adult female and male rats. Hepatic microsomes from male rats had the highest specific activity: 42.3 +/- 6.0 nmol SA mg(-1) min(-1) (mean +/- SEM). Kidney, intestine, stomach and serum activities were 60, 30, 14 and 0.7% with regard to the liver. In contrast, gastric microsomes from female rats showed the highest specific activity: 53 +/- 22.1 nmol SA mg(-1) min(-1) (mean +/- SEM) whereas intestine, liver, kidney and serum activities were 60, 43, 40 and 1.7% with regard to the stomach mucosa. Hepatic, renal and intestinal microsomes had a pH optimum of 5-6. Male rats had Vmax and Km values of 95.5, 83.4 and 29.4 nmol SA mg(-1) min(-1) and 2.9, 1.27 and 6.4 mM, while for female rats they were 54.8, 75.8 and 59.4 nmol SA mg(-1) min(-1) and 2.6, 1.35 and 3.4 mM for hepatic, renal and intestinal microsomes, respectively. Parathion inhibited the hydrolysis of ASA with an IC50 of 1.2 x 10(-5) M for liver and kidney and 5 x 10(6) M for intestine from male rats.

Entamoeba histolytica: increase of enterotoxicity and of 53- and 75-kDa cysteine proteinases in a clone of higher virulence.

We compared the enterotoxicity and cysteine proteinases (CP) of the low-virulence Entamoeba histolytica HM1 strain with the highly virulent 1659 clone, derived from HM1 by hamster liver passages. Enterotoxicity of 50,000 freeze-thawed trophozoites was determined on 0.28-cm2 intestinal segments mounted in Ussing chambers; CP activity of Nonidet-P40 amebal lysates was assayed by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and carbobenzoxy-L-arginine-L-arginyl-p-nitroaniline, a CP-specific substrate. Treatment of gerbil cecum segments with amebal lysates caused an immediate fall of their electrophysiologic properties (potential difference, short-circuit current, and transmural resistance) whose decay rates were clearly faster with 1659 than with HM1 lysates. Nonimmune and immune antiamebic human sera and the CP-specific inhibitor E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) prevented the fall of the electrophysiologic properties. Gelatinases, less active in HM1 than in 1659 trophozoites, were better preserved in lysates containing 10 mM p-hydroxymercuribenzoate (pHMB) to prevent autoproteolysis: in lysates without pHMB nearly no gelatinase bands were observed in HM1 samples, whereas intense 30K, 35K, 44K, and 75K bands were seen in 1659 samples; in lysates with pHMB only 53K and 75K bands were found that were much more intense in 1659 samples, 75K being barely visible in HM1 samples. The overall CP activity was 17 times higher in 1659 than in HM1 lysates, was inhibited by E-64 (mean inhibitory dose, 20 microM), was stimulated by 2-mercaptoethanol (ME) 3.7 times in HM1 and 2.4 times in 1659 lysates, and was reactivated by ME in lysates containing pHMB. Most of the CP activity in HM1 lysates sedimented at 15,600g but predominated in 1659 supernatants. The increase of E. histolytica virulence thus correlates with a remarkable increase both of in vitro enterotoxicity and of two CPs (53K and 75K), suggesting that these proteinases are significant pathogenicity factors.

Effects of intrauterine exposure to parathion on the activity of renal ATPases in offspring.

The effects of ethyl parathion on the activities of various renal enzymes were studied in the offspring from dams treated with this insecticide during pregnancy. The enzymes tested were the (Na+-K+)- and the Mg2+-dependent ATPases, the glutathione S-transferases and carboxylesterases. The postnatal effects of parathion on kidney ATPases from undernourished rats were also assessed. The organophosphate was administered per os to pregnant rats at a dose of 1 mg kg-1 body weight per day throughout gestation, and suspended after delivery. The offspring were divided in groups of normally-fed and undernourished rats. In the undernourished group, food restriction produced a decrease of 43% in body weight as compared to the normally-fed group. Offspring were sacrificed 6 weeks after birth and the enzymatic activities were determined in kidney homogenates. We found a decrease in the enzymatic activity of total ATPases, at the expense of the Mg2+-dependent ATPase. However, the activities of the (Na+-K+)-dependent ATPase, the glutathione S-transferases and the carboxylesterases did not show significant changes. On the other hand, undernutrition did not potentiate the effects of parathion on the ATPases. Thus, this organophosphate administered during pregnancy produced a selective inhibition on the renal Mg2+-dependent ATPase from offspring, which was not potentiated by our undernutritional model.

Effects of subchronic parathion administration on sodium salicylate excretion kinetics in female rats.

Organophosphorus (OP) pesticides are considered to be environmental contaminants, and chronic exposure to low levels through the diet may affect drug action. To study this possible interaction, ethyl parathion was administered by intubation to female rats for 35 consecutive days at a dose of 0.05 or 0.2 mg/kg of body weight per day. At 7, 21 and 35 days after parathion was initiated, rats were administered a single dose of 20 mg/kg sodium salicylate intraperitoneally. Total salicylates, salicylic acid (SA), salicyluric acid (SU) and gentisic acid (GA) were determined in urine. At 7 days, parathion treatment slowed the excretion of total salicylates. This effect was more evident at longer treatment times. Total excretion of SA was increased at the expense of GA at 7 days. However, this effect was reversed at 21 and 35 days. Excretion of SU was drastically diminished after 21 days of treatment with parathion. The results suggest that subchronic oral administration of parathion to female rats changes the excretion kinetics of sodium salicylate through combined effects on renal excretion mechanisms and biotransformation processes. Thus, exposure to low concentrations of environmental contaminants may produce important changes in drug action.

Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus.

An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).

Biotransformation of organic nitrate esters in vitro by human liver, kidney, intestine, and blood serum.

The biotransformation of glycerol trinitrate (GTN), isosorbide dinitrate (ISD), pentaerythritol tetranitrate (PETN), erythritol tetranitrate (ETN), and mannitol hexanitrate (MHN) by extracts from human liver, small intestine mucosa, kidney, and blood serum was investigated. The glutathione-dependent organic nitrate ester reductase activity of the intestinal mucosa was 21, 4, 4, and 2 times higher than the liver activity for ISD, PETN, GTN, and ETN, respectively. The liver enzymatic activity for MHN was 35% higher than the intestinal activity and 56% higher than kidney enzyme activity. The order of increasing enzymatic rates was: ISD = PETN less than GTN less than ETN less than MHN in the intestinal mucosa; ISD less than PETN less than GTN less than ETN less than MHN in the liver; and ISD less than PETN = GTN less than ETN less than MHN in the kidney. Human serum also metabolized these organic nitrates at lower rates than the studied organs. Thus, the serum specific activities were 1/5 for MHN, 1/30 for ETN, 1/40 for GTN, 1/44 for ISD, and 1/2000 for PETN of the activity present in kidney. On the other hand, the activity of human albumin was lower than that of blood serum. The serum and albumin activities were not modified by reduced glutathione or sulfhydryl inhibitors. These results suggest that small intestine may play an important role in the biotransformation of these drugs at their absorption site, after oral administration. They also demonstrate the possible participation of various human tissues in the overall metabolism of organic nitrate esters.