Acute effects of halothane and enflurane on drug metabolism and protein synthesis in isolated rat hepatocytes

The metabolism of sulphanilamide, antipyrine and paracetamol was studied in the absence and presence of the anaesthetics halothane and enflurane at three different concentrations (0.5, 1.0 and 2.0 mM) in isolated hepatocytes from the rat. Cell viability and protein synthesis were monitored to evaluate toxic effects. A strong concentration related inhibition of antipyrine oxidation (40-70%) and paracetamol conjugation (20-40%) was caused by both halothane and enflurane. Acetylation of sulphanilamide was not inhibited, however, as a slight augmentation was noticed. A significant dose related decrease of cell viability (3-13%) was caused by both anaesthetics. Dose dependent inhibition of the synthesis of stationary cell proteins (15-60%) and the synthesis/secretion of medium proteins (35-85%) was caused by halothane. Similar but slightly less pronounced effects were caused by enflurane. The present findings show that volatile anaesthetics may have general effects as well as different degrees of specific effects on both membrane bound enzyme and soluble enzyme activities.     

Inhibition of Myeloid Differentiation by Mature Granulocytes can be Reversed by Humoral Factors

Human mature granulocytes added to in vivo diffusion chamber (DC) cultures of murine bone marrow cells (BMC) led to a late reduction of CFU-C, day 5, and proliferative granulocytes, day 6. Cell free DC fluid harvested on day 4 from chambers with BMC and mature granulocytes slightly reduced granulocyte formation in new DC cultures, but not more than DC fluid from control cultures (BMC). Mice which had carried chambers of either group for 4 d, were not different when used as hosts for new DC cultures. However, they were less able to stimulate cell growth in DC than untreated mice. Transfer of DC to new host mice on day 2 completely reversed the inhibition by mature granulocytes which otherwise could be detected in numbers of CFU-C on day 5 and proliferative granulocytes on day 7. However, CFU-S numbers were reduced on day 5 following reimplantation indicating an increased differentiation of CFU-S. Transfer of DC as late as day 4, rapidly restored the granulocyte numbers in the granulocyte co-culture group suggesting that also more differentiated granulocytes were stimulated to further growth and differentiation. It is proposed that factors from mature granulocytes reversibly inhibit myeloid differentiation. Differentiation can again be induced by factors which reach the DC shortly after intraperitoneal implantation in a mouse.     

Effect of an analgesic drug (paralgin forte) upon laboratory pain under different cognitive manipulations: An experimental study

The pain-reducing effects of paralgin forte (under double-blind conditions) were studied under 3 different experimental conditions: 1) neutral instructions; subjects were given some information about the kind of unpleasant experience/pain involved in the experiment, 2) positive instructions; subjects were informed that the experiment did not involve painful experiences, and 3) negative instructions; subjects were informed that the experiment involved relatively severe pain. Both positive and negative instructions produced significant pain-reducing effects. With regards to paralgin forte, neither pain-reducing effects nor significant differences between paralgin forte and placebo were found. There was no interaction effect between drug and instruction variables. It is concluded that the instruction factors were effective in pain reduction in this experiment.     

Effect of diethylether on the formation of paracetamol sulphate and glucuronide in isolated rat hepatocytes

Diethylether has previously been shown to inhibit several pathways of drug metabolism, including conjugation of paracetamol in isolated rat hepatocytes. Since overall paracetamol conjugation consists of pathways of different subcellular localization (cytosolar sulphation and microsomal glucuronidation) the response of both pathways to diethylether was tested. The elimination of paracetamol (160 mumol/l, initial concentration) and the formation of paracetamol sulphate and glucuronide were measured (high-performance liquid chromatography) in suspensions of isolated rat hepatocytes from fasted and fed animals over 1 h in the absence and presence of diethylether (30 mmol/l). Approximately 90% of the paracetamol elimination was by sulphation and nearly 10% by glucuronidation both in the controls and in the presence of ether. The overall disposition of paracetamol and the formation of sulphate were both reduced by about 50% in the presence of ether compared to the controls while the formation of glucuronide was reduced by 70%. The results were not influenced by the nutritional state of the animals before sacrifice. It is concluded that the inhibitory effect of ether on total paracetamol metabolism was mainly caused by reduced sulphation. Since microsomal glucuronidation was also inhibited by ether, both cytosolar and microsomal enzyme systems were sensitive to diethylether.     

Hamstrings and gastrocnemius co-contraction protects the anterior cruciate ligament against failure: An in vivo study in the rat

An anesthetized rat model was used to study the effects of muscle contraction on the ultimate tensile load and the energy absorption at failure of the anterior cruciate ligament. In both knees, the joint capsule and ligaments, except for the anterior cruciate ligament, were divided, and the menisci were removed with the aid of a stereomicroscope. The cruciate ligament of the right knee was tested in tension until failure by femorotibial distraction during contraction of the hamstrings and calf muscles induced by electrical stimulation of the ischiatic nerve. The cruciate ligament of the left knee, which was loaded to failure with nonstimulated (relaxed) muscles, served as the control. The mean ultimate tensile load during muscle contraction was 86 N compared with 53 N when tested with relaxed muscles (p < 0.001). The energy absorption at failure was 0.41 and 0.19 J during contraction and relaxation, respectively (p < 0.05). This study suggests that previous investigations evaluating the force and energy necessary to rupture the anterior cruciate ligament (with use of a femur-anterior cruciate ligament-tibia complex stripped of all soft tissues and without gastrocnemius-hamstring muscle contractions) are incomplete and probably not representative of the in vivo situation.     

A Reinvestigation of the Usefulness of Breath Analysis in the Determination of Blood Acetaldehyde Concentrations

Human volunteers were given ethanol (0.4 g/kg) either intravenous or per os. They were also given ethanol (0.2 g/kg) intravenous 4 hr after receiving a dose of 50 mg titrated calcium carbimide, an aldehyde dehydrogenase inhibitor. During the first hour after starting the administration of ethanol, ethanol and acetaldehyde concentrations were determined in expired air, blood from the right atrium, arterial blood, and venous blood. In the absence of calcium carbimide treatment, the respective maximal blood acetaldehyde concentrations were (range): 6-30 μM (calculated from breath analysis using a Moodrbreath partition ratio of 190 for acetaldehyde); 0-3.5 μM (right atrium blood); and 0 μM (arterial and venous blood). After calcium carbimide treatment, the maximal blood acetaldehyde concentrations were 10-220 μM (calculated from concentrations in expired air), 38-280 μM (right atrium), 31-250 μM (arterial Wood), and 7-186 μM (venous blood). With aldehyde dehydrogenase inhibition, a clear correlation existed between breath concentrations and blood concentrations. Without this inhibition, no such correlation was found. A clear arterio-venous difference was seen for acetaldehyde concentrations whUe they were artificially elevated by calcium carbimide. Our study suggests that factors other than the equilibration of acetaldehyde between alveolar air and pulmonary blood are of great importance in determining the concentration of acetaldehyde in expired air.     

Covalent binding of 2,4-diaminoanisole and 2,4-diaminotoluene in vivo.

We have studied the activation of 2,4-diaminoanisole (2,4-DAA), a mutagenic hair-dye component, and 2,4-diaminotoluene (2,4-DAT), a hepatocarcinogen, to products which blind covalently to tissue macromolecules. Four hours after a dose of 100 mg/kg ring-labeled 3H-2,4-DAA, 0.30 nmol is found covalently bound per mg liver protein. This amount is increased by 83% after phenobarbital pretreatment, and by 43% after beta-naphthoflavone-pretreatment. Almost the same degree of binding is seen in kidneys. Subcellular fractionation of livers shows that most of the bound material is in the microsomal fraction. Similar levels of covalent protein binding is seen after administering ring-labeled 3H-2,4-DAT. No significant binding to DNA in vitro or in vivo could be demonstrated using 3H-2,4-DAA or 3H-2,4-DAT, whereas 3H-2,4-DAT is found to covalently bind to hepatic RNA.     

Determination of glomerular filtration rate (GFR) by analysis of capillary blood after single shot injection of 99mTc-DTPA

^99mTc-DTPA was prepared from a kit produced by the Institute of Atomic Energy, Oslo, Norway. Radiochemical purity as determined with gel chromatography ranged from 98.5–99.7% (n=7). The radiopharmaceutical showed no red cell uptake and not more than 0.2% protein binding in in vitro biokinetic studies.The clearance of ^99mTc-DTPA was compared to the clearance of ¹²⁵I-Iothalamate simultaneously using single shot intravenous injection and biexponential analysis of plasma activity disappearance rate according to Sapirstein et al. (1955). ¹²⁵I-Iothalamate was found to have a higher second volume of distribution than ^99mTc-DTPA, but there was no statistically significant difference in clearance.GFR calculated from capillary serum ^99mTc-DTPA count rates was in all subjects investigated virtually identical with GFR calculated from simultaneously collected venous plasma samples.Estimation of GFR on the basis of plasma activity curves obtained from sampling in two hours gave higher values than estimation from four hours sampling irrespective of kidney function and whether ^99mTc-DTPA or ¹²⁵I-Iothalamate was used.It is concluded that ^99mTc is almost entirely bound to DTPA after intravenous injection of the ^99mTc-DTPA complex, and that the complex is a suitable agent for determination of glomerular filtration rate, using both venous and capillary blood sampling.     

In Vivo Reversibility of the Jejunal Glucose and Cation Transport Alteration Caused by Intraluminal Surfactants in the Rat

Abstract Tied jejunal loops in anaesthetized rats were under standardized conditions pre-exposed for 30 min. with Tyrode solution containing surfactants. 5, 20 or 150 min. after wash out of bulk surfactant, the loops were re-instilled with Tyrode containing glucose at 5–15 mmol/l. Net glucose, sodium and potassium transport were studied for 15 min. by changes in intraluminal amounts, and compared with results obtained in control rats. The surfactants (mmol/l) tested were the anionics dioctylsulphosuccinate (5.6) and dodecylsulphate (8.5–17), the cationics cetrimonium bromide (2.1–4.1) and benzalkonium chloride (2.1), the nonionics Triton X100 (0.25%) and Lubrol WX (0.25–0.5%) plus cholic acid (4.9) and desoxycholic acid (1.3–2.5). In most cases, the glucose transport was normal or fairly normal after 150 min., most of the restoration taking place shortly after surfactant removal. However, Lubrol in particular caused more irreversible effects. Generally, the changes in net cation transport tended to be less easily reversible than the alteration in glucose transport. In so far as a normal or near to normal glucose transport is unlikely to occur unless both functional and structural integrity of the epithelium is preserved, the results indicate that in most cases there is but insignificant epithelial damage under the experimental conditions. Since, furthermore, these surfactants can interact with glucose transport in the same technique even at lower concentration and shorter incubation time than used here, it is concluded that the interaction of surfactants with intestinal transport is not neccessarily linked to gross histo-pathological changes.     

p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine

This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, gammaH2AX reverses after camptothecin removal. By contrast, gammaH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21(CDKN1A) (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced gammaH2AX formation in p21-/- cells, we conclude that enhanced gammaH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced gammaH2AX formation and inhibits camptothecin-induced p21(CDKN1A) upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that gammaH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21(CDKN1A) prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.