ROYAL ACADEMY OF MEDICINE IN IRELAND SECTION OF BIOMEDICAL SCIENCES Proceedings of Summer Meeting held 22nd & 23rd June, 1993. 2. Seuwen, K., Lagarde, A., Pouysseger, J. Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide specific phospholipase C. EMBO J. 1988; 7: 161-168.
ENDOTHELIN SIGNALLING IN PULMONARY ARTERY SMOOTH MUSCLE CELLS A. Kent and A. K. Keenan. Department of Pharmacology, University College Dublin, Belfield, Dublin 4.
COMPARISON OF THE ABILITY OF CALCIUM CHANNEL BLOCKERS AND PHOSPHODIESTERASE INHIBITORS TO RESTORE CARDIAC OUTPUT FOLLOWING INHIBITION OF NITRIC OXIDE SYNTHESIS
Endothelin (ET) is thought to mediate its constrictor actions in the vasculature via more than one signal transduction pathway (~). In this study we examined the coupling of ET-1 to phosphatidylinositol (PI) turnover and to uptake of 4SCa in bovine pulmonary artery smooth muscle cells. Cells were cultured from bovine pulmonary arteries by the explant method and smooth muscle identity confirmed using a fluorescent antibody to smooth muscle actin. ET-1 stimulated PI turnover was monitored as the accumulation of [3H]-inositol phosphates according to Seuwen et al (2). For determination of 4SCa-uptake, cells were incubated with 5 I.tCi/ml isotope + ET-1. Accumulation of [3H]-inositol phosphates was stimulated by 1 nM ET-1 in a time dependent manner with a 2 fold increase recorded at 20 min. While [3H]-InsP1 levels increased steadily over the period, transient rises in [3H]-InsP2 and [3H]-InsP3 were observed. 4SCa-influx into cells was stimulated in a concentration and time dependent manner (maximal effect at 1 I.tM ET- 1). In conclusion the differing concentration dependances of ET1 stimulated PI turnover and 45Ca-influx would suggest that these events are mediated independently in pulmonary artery smooth muscle cells: Supported by EOLAS and thc Irish Heart Foundation.
*N. A. Herity, **J. D. Allen, **~'B. Silke, and *A. A. J. Adgey. From the *Regional Medical Cardiology Centre, Royal Victoria Hospital and Departments of **Physiology and ***Therapeutics and Clinical Pharmacology, Queen's University, Belfast, Northern Ireland.
Endothelium-derived nitric oxide (NO) is a vasodilator acting via a cyclic GMP mechanism. NO synthase inhibition restores blood pressure in septic shock but excessive inhibition increases mortality partly due to a fall in cardiac output. We have used an NO synthase inhibitor - N~-nitro-L-arginine methyl ester (L-NAME) - to investigate this toxicity in the pentobarbitoneanaesthetised pig (Sagatal, M&B 30 mg/kg). Systemic (SVR) and pulmonary (PVR) vascular resistance and cardiac output (CO) were measured before and after administration of a bolus of L-NAME (10 mg/kg i.v.) followed either by nicardipine (NIC 0.05 -0.2 mg/kg i.v.: n=8), theophylline (THEO 7.5 - 10 mg/ kg i.v.: n=8) or the cGMPspecific phosphodiesterase inhibitor M&B 22948 (M&B 1 - 5 mg/kg i.v.: n=8). Results are expressed References in the table. Systemic vasoconstriction following L-NAME was 1. Masaki, T., Kimura, S., Yanagisawa, M., Goto, K. Molecular reversed by nicardipine and partly reversed by theophylline and cellular mechanism of endothelin regulation - Implications for associated with complete and partial restoration of cardiac output vascular function. Circulation. 1991; 84:1457-1468 respectively. All three vasodilators partly reversed pulmonary vasoconstriction. Depression of cardiac output following inhibition of NO synthesis is a reversible effect of increased afterload on the heart. These findings may prove valuable in the treatment of septic shock. Table Pre-Rx L-NAME NIC Pre-Rx L-NAME THEO Pre-Rx L-NAME M&B SVR 701 1591" 507t 1079 2369* 1994t 834 1783" 1951 PVR 154 406* 178t 222 527* 316t" 150 339* 203"I" CO 7. I 5.8* 8.61" 5.5 4.1 * 4.7~f 6.7 5.0* 5.0 SVR and PVR expressed in dyne.sec/cm5. CO in Vmin. Pre-Rx=Pre-treatment value. *P<0.01 vs control group, tP<0.01 vs L-NAME value.
THE INFLUENCE OF COLD AIR, L-MENTHOL AND CO 2 ON VENTILATION AND LARYNGEAL RESISTANCE IN THE ANAESTHETISED RAT. K. D. O'Halloran. A. K. Curran and A. Bradford. Department of Physiology, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2. Upper airway (UA) CO 2 and cold air alter laryngeal receptor activity ~ and reflexly modify UA airflow and muscle activity.
Therefore, cold air and CO 2 may also influence laryngeal resistance (L~N) In 30 adult, male, anaesthetised (chloralose, 100 mg/kg and urethane, 1000 mg/kg I.P.) spontaneouslybreathing rats, a constant flow (20 ml/s/kg) of warm (36~ humidified air containing 1-menthol (a specific cold receptor stimulant) or 0, 5 and 9% CO 2 and cold (30~ room humidity air was delivered to the isolated larynx with intact recurrent and superior laryngeal 96
Vol. 163 No. 2 nerves (SLN). Laryngeal airflow, temperature and sub-glottic pressure and spontaneous tracheal airflow were recorded. LRNshowed typical oscillations in phase with breathing. CO 2 had no significant effect (Student's paired t-test, significance at p<0.05) on tracheal airflow or LRN. Cold air caused a large fall in LRN (-42.4+2.7% SEM of control) and a decrease in respiratory rate (-31.2 +4.1% SEM of control) but 1-menthol had no significant effects. SLN section abolished the ventilatory inhibition and significantlyreduced the LRr, I response (-23.6_+2.4% SEM of control) to cold air. We conclude that laryngeal cooling, but not CO 2 reflexly alters LRN. Reference
1. Bradford, A., Nolan, P., McKeogh, D., O'Regan, R. G. The responses of superior laryngeal nerve afferent fibres to laryngeal airway CO2 in the anaesthetized cat. Exp. Physiol. 1990; 75: 267-270. Supported by the Health Research Board and Royal College of Surgeons in Ireland.
DELAYED ONSET MUSCLE SORENESS: EFFECT OF COMBINED PHOTOTHERAPY/LOW INTENSITY LASER THERAPY AT LOW PULSE REPETITION RATES J. A. Craig, P. Barlas, G. D. Baxter, D. M. Walsh and J .M. Allen. Biomedical Sciences Research Centre (Departments of Occupational Therapy & Physiotherapy and Biological & Biomedical Sciences), University of Ulster, BT37 0QB, N. Ireland. A double-blind, placebo controlled study, using male subjects (n=48), was conducted to investigate the efficacy of two different frequencies of combined phototherapy/low intensity laser therapy on alleviating the symptoms of Delayed Onset Muscle Soreness (DOMS). The study was approved by the University's ethical committee. After screening for relevant pathologies, recent analgesic, steroid drug usage or current pain, subjects were randomly allocated to one of four experimental conditions: Control, Placebo, Treatment Condition 1 and 2 (660-950nm; 31.7 J/cm2; pulsed at 5Hz for Condition 1 and 20 Hz for Condition 2). DOMS was induced in the non-dominant arm, which was exercised in a standardised fashion until exhaustion, using eccentric contractions of the elbow flexors. The procedure was repeated twice to ensure exhaustion was achieved. Subjects returned on two consecutive days for further treatment and assessment. The range of variables used to assess DOMS included range of movement (universal goniometer), mechanical pain threshold/tenderness (algometer) and pain (visual analogue scale and McGill Pain Questionnaire). Measurements were taken before and after treatment on each day except for the McGill Pain Questionnaire which was completed at the end of the study. Analysis of results using repeated measures ANOVA and relevant post-hoc tests showed a significant (p>0.05) nocicebo effect when compared with Treatment Condition 2 and Control in respect of the tenderness values. No other significant differences were found. In keeping with our previous findingstu, these results provide no convincingevidence for the putative hypoalgesic effect of this modality upon delayed onset muscle soreness. Reference
1. Craig, J. A., Fegan J. A., Baxter G. D., Allen J. M. Effect of laser irradiation upon delayed onset myogenic pain evoked by eccentric exercise. Ir. L Med. Sci. 1993: (in press).
Section of Biomedical Sciences 97 A CYTOGENETIC ANALYSIS OF THE EFFECTS OF LOW LEVEL THERAPEUTIC LASERS ON FRIEND ERYTHROLEUKAEMIA CELLS I. D. Logan, Y. A. Wilkinson, and P. G.McKenna. Dept. of Biological and Biomedical Sciences, University of Ulster at Jordanstown, Newtownabbey, N. Ireland, BT37 0QB. Low level lasers have wide usage in a variety of clinical situations. There is no clinical consensus as to appropriate treatment regimes, and there is a paucity of information as to the biological effects or clinical effectiveness. To address the concern about possible deleterious effects we have looked at the potential DNA damaging effects of low level laser irradiation (LLLI) at 2 Joules/cm~, a dosage commonly used in clinical practice. The cell types used were the thymidine positive clone 707 Friend erythroleukaemia cell and its thymidine kinase deficient subclone 707BUF, which has been shown to have increased sensitivity to a wide range of physical and chemical mutagens. Following irradiation of exponentially growing cells the end points examined were metaphase arrested chromosome spreads and micronuclei frequency in cytokinesis blocked cells. The results of this investigation showed that there were no increases in chromosome or chromatid aberrations, nor in the micronuclei frequency above zero irradiated controls. No DNA damaging effect has therefore been demonstrated. Currently by the estimation of mutation frequency at two suitable genetic loci and the immunochemical measurement of single strand DNA breaks we are applying more sensitive indicators to the assessment of any LLLI induced DNA damage. The financial support of the DHSS N.I. for this investigation is gratefully acknowledged.
CLASSIC USSING CHAMBER: DILTIAZEM TRANSPORT ACROSS RAT COLON EPITHELIUM D. J. Brayden, J. Dunne*, A. W. Baird* & J. G. Kelly. Elan Corporation. Drug Delivery Research Unit. Trinity College Dublin, Dublin 2. *Department of Pharmacology, University College Dublin, Belfleld, Co. Dublin. Our aim was test whether the Ussing chamber can be adapted for drug permeability studies across rat colonic epithelium using diltiazem as a model substance. Diltiazem was added to the apical side of the colon in concentrations ranging from 0.48 to 1.9mM. A range of 2 - 5% of drug was transferred to the basolateral side in 6 hours, but there was loss of 40 - 50% diltiazem from the donor apical side (n=18). The apparent permeability coefficient (P App) was calculated to be 2.81 X 10-6cm/sec. Losses from the apical side could be corrected by bathing in Krebs-Henseleit at a pH of 5.9, giving a new P App of 3.62 X 10-6 cm/sec (P<0.01). At pH 7.4 but not 5.9, diltiazem bound to Tygon| tubing. Flux of diltiazem was unaffected by the tight junction disrupter, cytochalasin D (3.71 gM), despite a mean reduction of transepithelial resistance of 60% in 1 hour (n=4). By comparison, cytochalasin D boosted absorption ofsalicyclic acid across rat colon (P < 0.02). Salicylic acid transport was concentration-dependent and gave a P App of 3.68 X 10-5 cm/sec. Unlike diltiazem, salicylic acid did not bind significantly to Ussing chamber components. Our experiments indicate that certain drugs includingdiltiazem bind to some surfaces of the Ussing chamber system. If such
98 Royal Academy of Medicine in Ireland
-
a problem arises, it may be countered by selecting from the newer modified single surface acrylic chambers tt~. Reference
1. Sutton, S. C., Forbes, A. E., Cargill, R., Hochman, J. H., & LeCluyse, E.L. Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial resistance using Sweetana-Grass diffusion cells. Pharm. Res. 1992: 9, 316-319.
POTENTIATION OF THE NMDA RECEPTOR EPSC IS INHIBITED BY METABOTROPIC GLUTAMATE RECEPTOR ANTAGONISTS J. J. O'Connor, M. J. Rowan and R. Anwyl.* Department of Pharmacology and Therapeutics and Department of Physiology*, Trinity College, Dublin 2. Previous studies have shown tetanically induced long-term potentiation (LTP) of N-methylD-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-epscs). In the present studies, we have investigated the action of the mGluR antagonists, L-2-amino-3phosphonoproprionate (L-AP3) and (RS)-amethyl-4-carboxyphenylglycine (MCPG) on tetanically induced LTP and aminocyclopentane-lS,3R-dicarboxylic acid (ACPD) enhancement of the NMDA-epsc in rat dentate gyrus granule cells, using the whole-cell variant of the patch clamp technique as previously described to the Society tl). NMDA-epscs evoked in response to stimulation of the proximal medial perforant pathway were isolated by the perfusion of 4-10 llM 6-cyano7-nitroquinoxaline-2,3-dione (CNQX) and picrotoxin (100 ~tM). L-AP3 (200 ~M) and MCPG (400 I.tM) both strongly reduced the amplitude and duration of the ACPD induced enhancement of the NMDA-epsc. Thus in the presence of LAP3, the enhancement of the NMDA-epsc 15 min following ACPD application was 105+12% compared to 170+t3% in controls (P<0.05; n=5). Similarly in the presence of MCPG, the potentiation of the NMDA-epsc after ACPD application was significantly reduced (e.g at 10min 97+6% versus 169+12% in controls; P<0.05; n=4). L-AP3 (200 ~tM) and MCPG (400 ~tM) also strongly antagonized the amplitude and time course of the tetanically induced potentiation. The potentiation of the NMDA-epsc in L-AP3 5 min following tetanic stimulation was 115+9%, of control, a significant reduction of 59% (P<0.05; n=5). Tetanic stimulation in the presence of MCPG completely blocked LTP, leaving only a very small amplitude short-term potentiation lasting 2 min. These results demonstrate that activation of metabotropic glutamate receptors by neurally released transmitter underlies LTP of the NMDA receptor mediated epsc. References
1. O'Connor, J. J., Rowan, M. J. & Anwyl, R. Whole-cell patch recording of excitatory synaptic transmission in rat hippocampal slices. Ir. J. Med. Sci. 1992; 161: 648.
THE EFFECTS OF TRIMETHYLTIN ON BEHAVIOUR AND LEVELS OF NITRIC OXIDE SYNTHASE IN THE BRAIN AND PERIPHERAL BLOOD NEUTROPHILS OF THE RAT M. Caldwell. B. Earley and B E. Leonard. Pharmacology Department, University College, Galway. Nitric oxide (NO) accounts for the biological properties of endothelium derived relaxing factor and is an endogenous stimulator of the soluble guanylate cyclase linking stimulation of glutamate receptors to changes in cGMP via brain NO synthase. NO is
I.J.M.S.
February, 1994 synthesized from the amino acid L-arginine by an enzyme, nitric oxide synthase (NOS). Trimethyltin (TMT), a neurotoxic organometal produces damage primarily in the CA3 and CA4 regions of the hippocampus, t~) Aims of this study: 1. To examine the effects of a single injection of TMT (6, 7 and 8 mg/kg i.p.), on locomotor activity 7 and 21 days post administration and its effects on Morris water maze and passive avoidance behaviour after 22 days. 2. To compare the effects of TMT on brain and peripheral blood neutrophil NOS levels 7 and 35 days post administration. Assay for nitric oxide synthase (NOS) NOS activity was measured by monitoring the conversion of [3HI arginine to [3HI citrulline. ~2) Changes in locomotor activity were observed 21 days post TMT at a dose of 8 mg/kg. TMT animals showed a significant deficit in Morris maze performance and passive avoidance behaviour at a dose of 8 mg/kg. NOS was induced in a dose dependent manner in neutrophils after 7 and 35 days. An increase in brain NO was observed in a dose dependent manner after 35 days only. This study confirms that TMT impairs spatial navigation and passive avoidance behaviour in rats indicating a role for the hippocampus in cognitive function. It is an attractive hypothesis to suggest that NO plays an important role in the neurodegeneration associated with TMT. References I. Bouldin, T. W., Goines, N. D., Bagnell, C. R., Krigman, M. R. Pathogenesis of trimethyltin neuronal activity; ultrastructural and
cytochemical observations. Am. J. Path. 1981: 104: 237. 2. Bredt, D. S., Sydner, S. H. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA. 1990: 87: 682-685.
BEHAVIOURAL EFFECTS WITH CRANIAL LOW LEVEL LASER IRRADIATION IN RATS ARE FREQUENCY DEPENDENT P. M. Wedlock, R. A Shephard and P. J Bracken. Dept. of Psychology, University of Ulster at Jordanstown, Newtownabbey, BT37 OQB. Recently it has been shown that high frquency (5KHz) cranial laser irradiation in rats can produce hyp6algesia. However this effect was short lived and reached only half the potency of morphine in the same paradigm ~1~. Findings from cellular, neurophysiological and clinical laser studies suggest that both the magnitude and direction of laser mediated analgesia may be frequency dependent. The aim of the present study was to determine the effects of four different frequencies of cranial laser irradiation using hot the plate assay. 17 rats received sham, 2.28 Hz, 18.24 Hz, 292 Hz and 5 KHz laser (Omega Laser Systems, London; 820nm; 6 + 1.5 J/cm) in a random order. Laser was applied to the rats' skulls immediately before exposure to the hot plate and hind paw lick latencies were recorded immediately, 15 mins, 30 mins, 60 mins and 24 hrs post laser administration. Analysis of Variance showed that the three lower frequencies produced significant hypoalgesia while the highest frequency (5KHz) produced significant hyperalgesia. This suggests that behavioural effects produced by laser irradiation of the CNS are frequency dependent. Reference
1. Wedlock, P. M., Shephard, R. A, Allen, J. M, and Baxter, G. D. Analgesia with cranial laser irradiation in rats is reversible with naloxone. Ir. J. Med. Sci; 1993; in press.
Section of Biomedical Sciences 99
Vol. 163 No. 2 CULTURED HUMAN SWEAT GLAND EPITHELIA: ISOLATION OF GLANDS USING NEUTRAL RED J. M. Fitzpatrick, J. G. Kelly & D. J. Brayden Elan Corporation. Drug Delivery Research Unit, Trinity College Dublin, Dublin 2.
disease and may provide a target for development of therapeutic agents. This work was supported by the Cystic Fibrosis Association of Ireland. Dr. R. A. Frizzell provided transfected CFPAC cells.
An improved sweat gland isolation method permits maximization of the yield of sweat glands without loss of cellular properties. Uptake of 0.5mM neutral red by sweat glands allowed a mean of 52% more glands to be visualized from the debris of chopped full thickness skin from 4 subjects. All glands were cultured as before tl). From 4 subjects a mean of 77% of unstained glands grew, by comparison with 70% from controls, (NS). Subcultured epithelia derived from both stained and unstained sweat gland cells gave similar electrophysiological features in Ussing chambers. For each group these included a transepithelial resistance range of 60-100 f2.cm 2 (n=4), a potential difference of -0.4 to -1.8mV (n=4) and a basal short-circuit current (Isc) of 5-15 I.tA.cm2 (n=4). Carbachol increased Isc by 10-20 I.tA.cm-2 (n=4), while amiloride reduced basal and abolished stimulated current (n=4). The absorption of nicotine (lmM) was similar in cells derived from stained glands. The permeability coefficient for nicotine absorption was 1.29 X 10-scm/sec in monolayers derived from neutral red stained glands (n=3) and 1.34 X 10.5 cm/sec in control monolayers (n=3). Neutral red staining thus improves the yield of sweat glands that can be isolated from skin. It does not alter the macroscopic ion and drug transport characteristics of the epithelia and therefore should be of henefit to sweat gland research.
1. Cliff, W. H., Shoumacher, g. A., Frizzell, R. A. cAMP-activated C1 channels in CFTR transfected cystic fibrosis pancreatic epithelial cells. Am. J. Physiol. 1992; 262: Cl154-Cl160.
Reference
1. Brayden, D. J., Cuthbert, A. W. & Lee, C. M. Human eccrine sweat gland epitheliai cultures express ductal characteristics. J. Physiol. 1988: 405, 657-675.
CHLORIDE EFFLUX IN CFTR-TRANSFECTED AND CYSTIC FIBROSIS PANCREATIC EPITHELIAL CELLS C. O'Reilly, E. Quinn, M. P. Ryan. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. Cystic Fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Loss of cAMP regulated chloride (C1) transport appears to be the basis for the CF defect and is a target for the development of therapeutic strategies. Pancreatic adenocarcinoma cells derived from a patient with CF (CFPAC-1 cells) and CFPAC-1 cells transfected with wild type CFTR were used ~ These cells were loaded with 36C1 for 2 hrs at 37~ Efflux was expressed as a % of total 36C1present in cells before efflux commenced and presented as mean + s.e.m (n=3). After 10 minutes, basal C1 efflux in CFPAC-1 cells was 31.2 + 3.8% and efflux from transfected cells was similar. Forskolin (10,I.tM) significantly (p<0.05) increased C1 efflux from the transfected ceils to 69.3 + 1.6% but had no effect on C1 efflux from CFPAC-1 cells. Bumetanide (100txM) and DIDS (100p.M) demonstrated some inhibitory effects on C1 efflux. Adenosine antagonists had no significant effects on chloride transport. Thapsigargin (0.5ktM) increased C1 efflux from both cell types. The results indicate that most of the C1 efflux under these conditions is most likely via CFTR. The response to thapsigargin indicates that calcium dependent CI efflux is unaffected in this
Reference
NOMINALLY CALCIUM-FREE BATHING SOLUTION HYPERPOLARISES RAT SKELETAL MUSCLE. J. O'Neill, R. P. Kernan, C. D. Craven. Department of Human Anatomy and Physiology, U.C.D., Dublin 2. Resting membrane potentials in superficial fibres of rat extensor digitorum longus muscles were measured in vitro at 29~ using 3M KCI back filled glass microelectrodes in solutions of normal Krebs and Krebs solution from which calcium had been omitted. At least 30 minutes was allowed for each muscle to equilibrate before measurements were made. Resting mean membrane potentials in normal and nominally calcium-free bathing solutions were -76.2+0.5 mV (225 penetrations) and -82.5+0.5 mV (215 penetrations) respectively, indicating a significant (p<0.01) hyperpolarisation of 6.3 mV on exposure to the nominally calcium-free bathing solution. On return to normal Krebs solution, the mean membrane potential fell to-77.9-+0.6 mV (97 penetrations) within 30 minutes. Addition of 10-4 M Ouabain to muscles bathed in calciumfree Krebs reversed the hyperpolarisation within 30 minutes. Low extracellular calcium increases the permeability of mammalian cardiac muscle to sodium~~ It is proposed that a similar increase in membrane permeability occurs in skeletal muscle and that the resultant influx of sodium stimulates the sodium-potassium pump, which, because of its electrogenicity, hyperpolarises the cell. Alternativelyan outward potassium current via sodium activated potassium channels~2~could push Em towards its equilibrium potential of-89 mV, resulting in the observed hyperpolarisation. (This study was supported by the Health Research Board of Ireland.) References
1. Rodrigo, G. C., Chapman, R. A. The calcium paradox in isolated guinea-pig ventricular myocytes: effects of membrane potential and intracellular sodium. J. Physiol. 1991: 434, 627-645. 2. Rodrigo, G. C., Chapman, R. A. A sodium activated potassium current in intact ventricular myocytes isolated from the guinea-pig heart. Exp. Physiol. 1990: 75, 839-842.
EFFECTS OF CYCLOSPORIN A ON VIABILITY AND INTRACELLULAR CALCIUM IN LLC-PK 1 AND MDCK CELLS E. Healy, H. Clarke, M. P. Ryan. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. Cyclosporin A (CsA) is a potent immunosuppressive drug whose use is limited by nephrotoxicity. We have investigated the time and dose dependency of changes in cellular viability and alterations in free intracellularcalcium [Ca]i induced by CsA in the established renal epithelial cell lines MDCK (of distal
100 Royal Academy of Medicine in Ireland tubular/collecting duct origin) and LLC-PK1 (of proximal tubular origin) . Cellular viability was assessed using the neutral red cytotoxicity assay following treatment for 4 to 24 hours with doses of CsA ranging from 4.2nM to 831.tM. Calcium was measured using the calcium - sensitive fluorescent probe Indo-lAM in a BD Facstar Plus Flow Cytometer. A dose and time dependent loss of cellular viability was seen in both cell types for doses of CsA ranging from 4.21xM to 83~M.No significant loss in cellular viability was seen at doses of CsA less than 4.2~tM. At 4 and 6 hours the LLC-PK1 cells exhibited a greater loss of viability compared to the MDCK cells. Resting [Ca] i was found to be 174 + 50nM in LLCPKI cells . CsA treatment for 24 hours increased the resting [Ca]~by 107+13, 128+10, and 226+67nM at doses of 4.2nM, 42nM and 4.2l.tM CsA respectively. The greater susceptibility of LLC-PKI cells to CsA induced damage indicates a possible site selective action of CsA along the nephron. Alterations in [Ca]i may play an important role in the mediation of its toxicity. This work was supported by EOLAS and the E.C. BRIDGE Programme. Cyclosporin A was a gift from Sandoz Pharma. Ltd.
COMPARATIVE NEPHROTOXIC1TY AND CYTOTOXICITY OF CISPLATINMETHIONINE COMPLEXES
I.J.M.S.
February, 1994 HYPERGLYCAEMIA INDUCES EXTRACELLULAR AND INTRACELLULAR GLYCATION OF INSULIN Y. H. A. Abdelwahab, C. R. Barnett, P. R. Flatt. Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, BT52 ISA. Glycation of insulin following exposure to hyperglycaemic concentrations of glucose was determined using crystalline insulin and extracts of clonal insulin-releasing RINm5F ceils maintained in tissue culture. After dialysis to remove free glucose, insulin was separated into glycated and non-glycated fractions by affinity chromatography using Glycogel B prior to measurement by radioimmunoassay. Incubation of insulin (7 ~tmol/l) in phosphate buffered saline (pH 7.4; 37~ at a range of glucose concentrations (5.6-220.0 mmol/l) for 1-48 hours revealed a time and concentration-dependent increase of insulin glycation. Maximum glycation of insulin was 22.3 + 2.4% (mean + SEM, n=3), but significant glycation occurred within 8-20 hours of incubation at 14.0-21.0 mmol/l glucose. Glycation was decreased by 70% at 4~ compared with 37~ (P<0.02), and enhanced 2.2-fold (P<0.001) by an increase of acidity from pH 8.4 to 6.5. Culture of RIMm5F cells in RPMI-1640 for 72 hours with 11.1 mmol/1 glucose resulted in a 3.2-fold increase of insulin glycation (10.9 + 1.5% vs 3.4 + 2.1% at 5.6 mmol/1 glucose; n=6; P<0.01). These observations suggest that circulating glucose concentrations encountered in diabetes result in the glycation of insulin within the pancreatic B-cells. Secretion of glycated insulin with reduced biological potency may contribute to the insulin resistance of diabetes.
C. A. Nolan, E. Healy, C. O'Connell, P. M. Deegan, M. P. Ryan. Department of Pharmacology, University College Dublin, Belfield, Dublin 4. Cisplatin (CP) is a very effective antineoplastic agent whose clinical use is limited by nephrotoxicity. We have previously shown that a CP-methionine complex at a ratio of 1:5 (w/w) did not produce nephrotoxicity in vivo compared to CP alone while retaining some antimitotic activity in vitro~lL We have now extended these studies to a wider range of CP-methionine ratios with the aim of retaining full cellular antiproliferative activity with significant reduction in nephrotoxicity. The relative nephrotoxicity in vivo of CP and CP-methionine complexes (1:0; 1:0.2; 1:0.5; 1:1 and 1:5) were assessed in male Wistar rats 5 days after i.p. administration of 6 mg CP/kg body weight. CP caused significant elevations (p<0.001) in blood urea nitrogen (BUN) and plasma creatinine levels indicating nephrotoxicity. Animals treated with CPmethionine complexes demonstrated protection from nephrotoxicity. Kidneys from CPmethionine treated rats indicated a lesser degree of structural damage to the proximal tubule. In the C6 glioma cell line, [3n]thymidine incorporation fell to less than 5 % of control following CP (51.tg/ml) treatment. Incubation of cells with CP-methionine at 1:0.2; 1:0.5 and 1:1 ratios also resulted in very significant cytotoxic effects. These results indicat e that CP-methionine complexes at ratios which were highly cytotoxic in vitro exhibited very little nephrotoxicity and suggest a possible role for either methionine co-treatment or platinum-methionine compounds in cancer treatment. This work was supported by Eolas. Reference
1. Ormond, T, Pratt, I. & Ryan, M. P. Reduced nephrotoxicity in vivo and in vitro of cisplatin-methionine complex. Br. J. Pharmacol. 1988; 95: 584P.
EXAMINATION OF BEHAVIOURAL AND BLOOD BIOCHEMICAL PARAMETERS FOLLOWING STREPTOZOTOCIN-INDUCED DIABETES C. MacSweeney, J. P. Kelly and B. E. Leonard. Department of Pharmacology, University College, Galway, Ireland. Streptozotocin (STZ) is an antibiotic which destroys the B cells of the pancreas, thereby rendering the animal diabetic. Observed changes following STZ include hyperglycaemia, polydipsia, polyuria, increased serum corticosterone and serum enzyme levelstJL However, an appraisal of behavioural changes following STZ has not been investigated. The objective of this experiment was to examine the effects of a single injection of STZ (60mg/kg) in male Sprague-Dawley rats administered via 3 different routes: intraperitoneal (i.p.), intravenous (i.v.) and subcutaneous (s.c.). The following behavioural paradigms were examined 2 weeks following STZ treatment: 1) "Open field", a measure of locomotor activity. 2) Plus maze, a measure of anxiety. 3) Morris maze, a measure of reference memory. Twenty eight days following STZ, rats were sacrificed and corticosterone as well as glucose, alkaline phosphatase, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. The results demonstrate that the route of administration is important in the full development of STZ-induced diabetes with regard to hyperglcaemia, polydipsia and serum biochemistry: the s.c. and i.v. routes gave similar results. The i.p. is not satisfactory as less than 50% of rats develop hyperglycaemia.
Section of Biomedical Sciences 101
Vol. 163 No. 2 No significant differences were found in the three behavioural paradigms regardless of route of STZ administration. Reference
1. Tomlinson, K. C., Gardiner, S. M., Hebden, R. A., Bennett, T. (1992). Functional consequences of streptozotocin-induced diabetes mellitus, with particular reference to the cardiovascular system. Pharmacol. Rev. 44: 103-141.
THE EFFECT OF CO 2 AND COLD AIR ON SUPRAGLOTTIC UPPER AIRWAY RESISTANCE IN THE ANAESTHETISED RAT A. K. Curran, K. D. O'Halloran, A. Bradford. Department of Physiology, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin 2. Upper airway (UA) CO 2 modulates laryngeal receptor activity and reflexly excites the genioglossus muscle ~1), a major UA dilator muscle. Therefore, UA CO 2 may be expected to reflexly reduce UA resistance and thereby be important in the regulation of UA patency. In 18 adult, male, anaesthetised (chloralose, 100 mg/kg and urethane, 1000 mg/kg I.P.), spontaneously breathing rats, a constant flow (10 ml/s/kg) of warmed (36~ humidified air containing 0, 5 or 10% CO a and cold (26~ room humidity air was delivered to the supraglottic UA. Spontaneous tracheal airflow and UA temperature, airflow and pressure were recorded. CO 2 had no significant effect (Student's paired t-test, significance at p < 0.05) on any of the measured variables whereas cold air caused a substantial fall in UA resistance (-44+3.3 % SEM of control) which was significantly less marked (-33+3.1% SEM of control) following superior laryngeal nerve section. We conclude that UA CO 2 does not reflexly alter UA resistance in anaesthetised rats. UA cooling markedly reduces UA resistance, partly through a superior laryngeal nerve-mediated reflex. Reference
1. Nolan, P., Bradford, A., O'Regan, R. G., McKeogh, D. The effects of changes in laryngeal airway C02concentration on genioglossus muscle activity in the anaesthetized cat. Exp. Physiol. 1990; 75: 271274. Supported by the Health Research Board and Royal College of Surgeons in Ireland.
portal hypertensive rats (1.66+0.129, n=6) than in vessels from sham rats (0.59+0.079, n=6, P<0.001). Endothelium-dependent relaxations to ACh were not significantly altered by portal hypertension. Hence, portal hypertension alters the responsiveness of mesenteric arteries to NA.
THE SIGNIFICANCE OF ADENINE PHOSPHORIBOSYLTRANSFERASE FOR DNA EXCISION REPAIR IN FRIEND ERYTHROLEUKAEMIA CELLS A. A. Nelson, Y. A. Wilkinson, P. G. McKenna. Dept. of Biological and Biomedical Sciences, University of Ulster, Coleraine. N. Ireland. BT52 1SA. Adenine phosphoribosyltransferase (APRT) deficient Friend erythroleukaemia (FEL) cells, 707DAP8 and 707DAP10, are more sensitive to mutagenesis by methyl methanesulphonate (MMS) and have significantly decreased ATP levels, compared to wild-type (APRT positive) cells3 I~ The increased mutagen sensitivity of these cells may be due to a deficiency of ATP for the activity of ATP-dependent DNA excision repair enzymes. The abilities of the APRT deficient cells to carry out DNA excision repair following MMS treatment was assessed using unscheduled DNA synthesis (UDS). Cells were treated with MM S (10l.tg/ml for 16hrs), radiolabelled and processed for the determination of UDS. The results showed that the APRT deficient ceils 707DAP8 and 707DAP10, had decreased levels of DNA excision repair (66% and 41% respectively, compared to wild-type). Further experiments were carried out using higher doses of MMS (1671.tg/ml for 1.5hrs). Cells were either radiolabelled immediately or after a 1.5hr recovery period. The results again showed decreased DNA excision repair in the APRT deficient cells. With no recovery period, 707DAP8 cells had decreased DNA excision repair levels, 74% of wild-type cells. With a 1.5hr recovery period 707DAP8 and 707DAP10 ceils had decreased repair levels, 77% and 28% respectively of wild-type cells. Therefore, APRT appears to have a significant role for DNA excision repair in FEL cells. The financial support of the Department of Education Northern Ireland and the Cancer Research Campaign are gratefully acknowledged. Reference
THE EFFECTS OF PRE-HEPATIC PORTAL HYPERTENSION ON VASCULAR RESPONSES IN RAT MESENTERIC ARTERIES
1. Amara, F. M., Wilkinson, Y. A., Ward, P. E., Thompson, C. C. M., McKenna, P. G. Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRTdeficient Friend erythroleukaemia cells. Mut. Res. 1991; 246: 151-155.
T. Cawley 1, J. Geraghty 2, H. Osborne z, J. R. Docherty I. ~Department of Physiology, RCSI, Dublin 2 and 2Department of Surgery, Beaumont Hospital, Dublin 9. We have examined the effects of pre-hepatic portal hypertension on the responsiveness of rat mesenteric arteries. Rats were made portal hypertensive by creating a portal vein stenosis, or sham operated. Effectiveness of surgery was assessed by measuring the degree of portalsysemic shunting using the radioactive microsphere technique. Degree of shunting was 59.6+7.2% (n=5) and 0.08+0.05% (n=5)in 14 day portal hypertensive and 14 day sham rats, respectively. Mesenteric arteries of diameter 200-350 l.tm were mounted in small vessel myographs for the investigation of contractions to noradrenaline (NA) and relaxations to acetylcholine (ACh). The maximum contraction to NA was significantly greater in vessels from
A DOUBLE BLIND INVESTIGATION OF THE HYPOALGESIC EFFECTS OF LOW PULSE REPETITION RATE H-WAVE THERAPY (2-16 HZ) UPON EXPERIMENTALLY INDUCED ISCHAEMIC PAIN B. C. McDowell, M. McCrory, G. D. Baxter, J. M. Allen, D. M. Walsh. Biomedical Sciences Research Centre (Departments of Occupational Therapy & Physiotherapy and Biological & Biomedical Sciences), University of Ulster at Jordanstown, Newtownabbey, BT37OQB. Previous studies at this centre have demonstrated the hypoalgesic effects of so called H-Wave Therapy (HWT; 60Hz),
102 Royal Academy of Medicine in Ireland an electrotherapeutic modality, upon experimentally induced ischaemic pain. The current study was similarly designed to investigate lower pulse repetition rates. This study was approved by the University's research ethical committee. Healthy human subjects (n-60; M=30 F=30) were recruited and screened for contraindications. Current pain was assessed by presentation of computerised visual analogue scales (VAS) and, on completion, McGill Pain Questionnaires (MPQ) were used to assess 'worst' pain experienced. Subjects were required to attend on two occasions. The first was used to establish baseline pain scores and at the second, 48 hours later, subjects were randomly assigned into one of five groups; 2Hz (n=12), 4Hz (n=12), 16Hz (n=12), Placebo (n=12) or Control (n=12). HWT was applied using two hydrogel electrodes and the current intensity increased until the subject reported a 'strong yet comfortable' sensation. Stimulation commenced 10 minutes prior to pain inductionand continued throughout the 12 minute duration of the technique. Analysis of VAS scores using ANOVA showed no significance between groups and, whilst analysis of the Pain Rating Index (PRI) showed a linear trend towards hypoalgesia, differences between groups proved not to be significant. Reference
1. Waish, D. M., Baxter, G. D., Allen, J. M., Bell, A. J., Mokhtar, B. (1992). An assessment of the analgesic effects of H-Wave therapy upon experimentally induced ischemie pain, lr. J. Med. Sci. 161,472.
NOVEL IMPLANT FOR THE LONG-TERM DELIVERY OF A GROWTH PROMOTING AGENT TO BEEF CATTLE P. B. Deasy, M. P. Finan, P. R Klatt*, Th. Hornykiewytsch*. Department of Pharmaceutics, Trinity College, Shrewsbury Road, Dublin 4. *Hoechst AG, Frankfurt am Main 80, Germany. Implants containing natural hormones such as oestradiol- 1715 are widely used outside the European Economic Community to aid weight gain and improve carcass quality in beef cattle. The novel extended-release implant described in this project is fully biodegradable and produces increasing delivery of active over 3-6 months duration to better mimic the dosage requirement than existing conventional or prolonged-release implants. The final implant developed was composed of a string of nine fiat-faced aligned compacts each 4 mm in diameter and 2 mm thick. The terminal compacts had reduced drug loading (50%) in the least biodegradable copolymer of polylactic/glycolic acid used (70: 30). Alternate compacts had higher drug loading (60%) in less biodegradable copolymer (80:20). These five active containing compacts were separated by four spacer inactive compacts, containing the most biodegradable copolymer used (60:40). The design minimized the initial burst of drug release and by progressively increasing the surface area of exposed active compacts gave long-term increasing release of oestradiol1713. The compact types and various implant designs were extensively evaluated in-vitro using radiolabeled drug and dissolution testing. The final implant and modifications thereof were also evaluated in steers, whose improved plasma profile of active and superior weight gain were demonstrated. The technology described can be modified for the variable delivery of other classes of drugs to humans or animals.
I.J.M.S. February, 1994 INVESTIGATION OF A POSSIBLE "APPETITE" FOR FIBRE IN ADULT SHEEP D. P. Campion and B. F. Leek. Department of Veterinary Physiology and Biochemistry, U.C.D., Veterinary College, Dublin 4. A physiological "drive" for roughages or fibrous foodstuffs, expressed clinically as hair-licking and crate-chewing, has been identified in liquid-fed veal calves"~. To discover if a similar drive may be identified in the adult ruminant, 3 sheep were housed in metabolic crates and fed a low fibre pelleted diet for a period of 8 days. Jaw movements were recorded for 24h periods to monitor rumination. Each sheep was offered a carton containing 50g polyethylene fibre, chopped into 10mm lengths, at the end of the 7th day. After 24 hours 21.2, 42.1 and 42.0g of fibre had been ingested and rumination was significantly (p < 0.05) increased (from 0, 0, 0, to 12, 179, and 177 min.day~). In a second experiment a carton containing 50g chopped polyethylene fibre was offered daily to 4 fistulated sheep immediately upon switching from hay to the pelleted diet. Fibre ingested increased significantly (p < 0.05) from 7.8 + 2.3g (mean + S.E.) at the end of day 1 to 15.4 + 2.3g at the end of day 4 and quantities of ruminated polyethylene particles were observed in the rumen fluid of each sheep. After roughages have been removed from the diet, several days of rumination and fermentation are required to reduce fibre retained in the forestomach. We believe that this increasing "fibre deficiency" may lead the animal to seek out fibre, even choosing otherwise unpalatable fibre, a possible causative factor in some foreign body pathologies and poisonings. Reference
1. Fox, M. W. Farm Animals, Husbandry, Behaviour, and Veterinary Practice. University Park Press, Baltimore 1984: 103.
RELATIONSHIP OF PERIPHERAL TO PERITONEAL VITAMIN E LEVELS IN WOMEN WITH MINIMAL STAGE ENDOMETRIOSIS AND A NORMAL PELVIS S. C. Sharma, C. Barry-Kinsella* and K. Foran. Department of Pharmacology & Therapeutics, Trinity College, Dublin 2 and *Department of Obstetrics & Gynaecology, RCSI, Rotunda Hospital, Dublin 1, Ireland. Women with minimal stage endometriosis (MSE) have below normal serum vitamin E "~ and recent studies in rats have shown that ovarian vitamin E is increased in the luteal phase and also when LH injection is given to them in this phase t2~.Since ovaries are the main contributors of fluid present in the Pouch of Douglas (POD) we have examined if POD levels of vitamin E in women with MSE are different from subjects with a normal pelvis and if a relationship exists between peripheral and POD values in the two groups. Following Ethics Committee approval and an informed patient consent 21 women with MSE and 19 controls in their luteal phase were recruited and laparoscopically assessed for the presence or absence of endometriosis. Samples of serum and peritoneal fluid were collected and stored at -20~ Vitamin E (t~-tocopherol) was analysed by HPLC and values shown are mg/1 (mean +.SEM). The results show that women vith MSE have serum and peritoneal vitamin E levels of 5.55 + 0.30 and 2.27 + 0.11 compared with normal group values of 6.45 + 0.41 and 2.66 + 0.15 respectively (p<0.05 for both comparisons). There is a correlation of serum and peritoneal
Vol. 163 No. 2
vitamin E levels in the two groups but the % age of vitamin which enters the POD in women with MSE is significantly lower than the normal group (37.70 + 1.57% vs 44.83%; p<0.05). It appears that a barrier exists to the entry of vitamin E from peripheral blood to the peritoneal fluid and this is disturbed in women with MSE. References
1. Sharma, S. C., Barry-Kinsella, C. and Foran, K. (1992). Serum vitamin E in patients with minimal stage endometriosis and a norma! pelvis. Irish J. Med.Sci. 161, 7. 2. Aten, R. F., Duarte, K. M., Behrman, H. R. (1992). Regulation of ovarianantioxidantvitamins,reduced glutathioneand lipidperoxidation by luteinizing hormone and prostaglandin F 2a. Biol. Reprod. 46, 401407.
Section of Biomedical Sciences
103
cells were studied using restriction fragment length polymorphism (RFLP) analysis. RFLP analysis of the aprt locus in the mutant cells revealed two very small deletions. One of these, B1, arose following irradiation of T K cells and the other D3B2 arose following irradiation of wild-type Friend cells. These deletions were less than 15 base-pairs in size and were mapped to a PstI fragment on the aprt locus between 2483-3100bp (spanning exons 4 and 5). One of the deletions B 1 was confinned by PCR and pinpointed to exon 4 (2483-2599bp). One other mutant B5 was identified following irradiation of this TK- cell line, the mutant had gained a restriction endonuclease (RE) site. The site was gained within an EcoRV fragment which spanned from 1091087bp of the aprt locus (includes exon 1). The financial support of the MRC is acknowledged.
INVESTIGATIONS OF PREJUNCTIONAL CONTROL OF NEUROTRANSMISSION IN RAT KIDNEY G. Thomas, J. R. Docherty. Department of Physiology, Royal College of Surgeons in Ireland, Dublin 2. Prejunctional o~2-adrenoceptors mediate a negative feedback inhibition whereby noradrenaline release is modulated by the level of activity. Little is known of the mechanism involved in this inhibition, although in some systems it may involve G proteins sensitive to n-ethyl-maleimide (NEM). We have investigated the effects of NEM on prejunctional inhibition in rat kidney slices. Rat renal cortex slices were pre-incubated for 30 rain with NEM (10-30l.tM) or vehicle and incubated for 30 min with 3Hnoradrenaline. Potassium chloride (KCI) evoked release of tritium was taken as a measure of transmitter release in superfusion experiments. In vehicle experiments, the c~2-adrenoceptor agonist xylazine and the c~2-adrenoceptor antagonist yohimbine decreased or increased KCI evoked release of tritium, respe ctively. Pre-incubation with NEM significantly reduced the effects of xylazine (10~tM) from 66+14% to-5+14% inhibition (P<0.01). Effects of yohimbine were not significantly altered. It is concluded that a G protein sensitive to NEM mediates the c~z-adrenoceptor mediated inhibition of neurotransmission in rat kidney. Supported by RCSI.
MOLECULAR ANALYSIS OF UV-INDUCED MUTATIONS AT THE ADENINE PHOSPHORIBOSYLTRANSFERASE LOCUS IN WILD-TYPE AND THYMIDINE KINASE DEFFICIENT FRIEND CELLS
THYMIDINE KINASE (TKI) GENE EXPRESSION AS MEASURED BY IN-SITU HYBRIDIZATION IN A HUMAN BREAST CANCER CELL-LINE (MCF-7) J. O'Connor, V. J. McKelvey-Martin, P. G. McKenna. Department of Biological and Biomedical Science, University of Ulster, Coleraine. Cytosolic TK (TK1) is a cell cycle regulated enzyme having its highest activity during DNA synthesis. Significant elevation of TKI activity has been reported in breast carcinoma relative to fibroadenoma and normal breast tissue; and this activity correlates positively with cancer recurrence. Numerous mechanisms at transcriptionaland translationallevels have been implicated in the control of TK activity. This communication presents a protocol for the in-situ detection of TK1 mRNA in individual cultured human breast cancer ceils (MCF-7) using an antisense TK1 mRNA probe. The protocol was verified using MCF-7 cells grown in medium supplemented with Tamoxifen or Estradiol 171~which have been shown by Northern hybridization to cause decreases and increases, respectively, in TK1 mRNA levels relative to untreated control ceils. Quantification ofTK1 mRNA hybridization was performed using an image analysis system and the expected changes in mRNA levels were observed. Further studies will evaluate the usefulness ofTK1 in-situ hybridization data in diagnosis and determining prognosis of breast cancer.
CELL CYCLE DELAY AND THYMIDINE KINASE ACTIVITY IN SYNCHRONISED FRIEND MOUSE ERYTHROLEUKAEMIA CELLS
M. W. McKinney, Y. A. Wilkinson and P. G. McKenna. Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, N. Ireland.
C. C. M. Thompson, P. J. McCarthy, V. J. McKelveyMartin, P. G. McKenna. Dept. of Biological and Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Northern Ireland, BT52 1 SA.
Thymidine kinase deficient (TK) Friend cells have been shown to be hypersensitive to a range of mutagens. The dCTP:dq'TP pool ratio showed an approximate 5-fold increase in TK- cells compared to wild-type cells. Total genomic DNA derived from UV induced (1.2-3.6 J/m z) adenine phosphoribosyltransferase (aprt) deficient mutants (20 in total) of wild-type and TK- Friend
Thymidine kinase (TK)-deficient subclones of mouse Friend erythroleukaemia cells, clone 707, are more sensitive than wildtype (wt) to DNA-damaging treatments, including shortwave ultraviolet (UV)-irradiation. Many DNA-damaging treatments also cause cell cycle delay which may increase time for damage repair.
I.J.M.S. February, 1994
104 Royal Academy of Medicine in Ireland
concentrations causing damage to cells and macromolecules ~2) This cellular damage will further promote inflammatory processes, including cell proliferation. Many ROS-induciblechanges are irreversible and so chronic disease processes may commence.
Delay induced by UV-irradiation of 707wt and TK-deficient subclone 707BUF following doses of 0, 1.2, 2.4 and 3.6 Jm z in the Gl-phase was determined. TK activity in 707wt around the cell cycle was measured both in untreated cells and in those irradiated at 2.4 Jm -~ in Gl-phase. Cells were synchronised in G~-phase using elutriation. Cell cycle stage was measured using flow cytometry. 707wt and 707BUF showed a similar overall delay period, mainly in S-phase, of approximately 2 hours for a dose of !.2Jm 2, 4-5 hours for 2.4Jm 2 and 9 hours for 3.6Jm 2. TK activity in untreated 707wt showed an increase with S-phase onset, declining as the population entered the next G-phase. Activity in irradiated cells followed the same initial pattern, but remained high during the delay period.
References
1. Palluy, O., Morliere, L., Gris, J. C., Borne, C., Modat, G. Hypoxia/ reoxygenation stimulates endothelium to promote neutrophil adhesion. Free Rad. Biol. Med. 1992: 13, 21-30. 2. Strain, J. J., Hannigan, B. M. and McKenna, P. G. The pathophysiology of oxidant damage. J. Biomed. Sci. 1991: 2, 19-24.
A ROLE FOR LUTEIN IN INFLUENCING OXIDATION IN LOW DENSITY LIPOPROTEIN (LDL). D . I . Thurnham*, M. Chopra* and D. Leake.** *Human Nutrition Research Group, Dept. Biology and Biomedical Sciences, University of Ulster at Coleraine, BT52 1SA. **School of Animal and Microbial Sciences, University of Reading, RG6 2AJ.
INFLAMMATION AND HEART DISEASE: WHICH IS CAUSE AND WHICH EFFECT? B. M. Hannigan. Department of Biological and Biomedical Sciences, University of Ulster at Coleraine, N. Ireland.
Lutein is one of the most common xanthophyll carotenoids (Ct) in our diet and is as abundant as B-carotene in most green and yellow vegetables. In the British population, plasma lutein forms 15% of the total Ct. Plasma lutein like ~-tocopherol (ctT) is correlated with plasma cholesterol in both sexes in contrast to the other main Ctt~L s T is the main antioxidant in lipoproteins (Lp) but does not correlate with the resistance of Lp to in vitro oxidation. We are investigating whether lutein and other Ct act synergistically with ctT in controlling the oxidation of Lp. In this report we describe the distribution of hydrocarbon and xanthophyll Ct in HDL and LDL. The distribution of Ct is similar in LDL and plasma. Likewise, the molar ratio of ctT : Ct is the same in LDL (x+SD, 11.7+13.6) and HDL (11.4+0.7) (table). However, the proportion of xanthophyll Ct was much higher in HDL than LDL; the lutein: B-carotene ratio alone was 4 to 20fold greater. We have shown elsewhere that lutein has better antioxidant properties than B-carotene~2~and high HDL is a negative risk factor for ischaemic heart disease. At the present time however the role of lutein in Lp is still being investigated.
Inflammation accompanying heart disease is frequently observed at autopsy. While this may indicate the onset of tissue healing, it is also possible that inflammatory processes can contribute to actual tissue damage. The current use of thrombolytic therapies may pre-dispose to the latter possibility. An important initiating event in inflammation is the recruitment of leukocytes to a tissue site. The expression of adhesion molecules, on leukocyte and endothelial cell surfaces, facilitates the capture of leukocytes from the circulation and their movement into the tissue. It has been shown ~) that adhesion molecule expression is significantly up-regulated within 30 minutes following tissue hypoxia and re-oxygenation (H/R). This effect is reduced by the presence of antioxidants such as superoxide dismutase and catalase. Possible effector molecules are therefore reactive oxygen species (ROS). Sources of ROS during H/ R and subsequent inflammation include the leukocyte respiratory burst, endothelial cells themselves, and the reaction catalysed by the enzyme xanthine oxidase, formed during hypoxia from xanthine dehydrogenase. In addition to their effects on adhesion molecules ROS, at low concentrations, can alter the expression of many genes in adjacent target cells with higher
References
1. Thurnham D. I. Lutein, cholesterol, and risk of cancer. Lancet 1989; ii: 441-42. 2. Chopra, M., Thurnham, D. I. In vitro antioxidant activity of lutein. In: Food and cancer prevention. London: Royal Society of Chemistry, 1992: in press.
TABLE Antioxidants ~tmol per g Lipoprotein-Nitrogen Subjects Total Carotene ct-Tocopherol s T : carotene
JR
PW
DL
LDL
HDL
LDL
HDL
GW LDL
HDL
LDL
1.44 12.88 8.9
0.27 2.88 10.5
0.90 13.85 15.4
0.15 1.86 12.2
1.07 15.13 14.1
0.36 4.11 11.3
1.61
13.27 8.22
HDL 0.1 4 1.62 11.8
Individual carotenoids as percentage of total carotene: B-carotene
16
8
6
ND**
33
16
12
7
Lutein & anhydrolutein*
19
43
23
62
20
45
9
39
* tentative identification; **' not detected
Section o f Biomedical Sciences
Vol. 163 No. 2 VITAMIN E-A NATURAL ANTIOXIDANT IN THE VASCULAR SYSTEM P. J. A. Sheehy. Department of Nutrition, University College, Cork. Vitamin E is an essential nutrient for all higher animals including man. Vitamin E activity in foods is derived from two groups of compounds of plant origin called tocopherols and tocotrienols. These compounds are present in varying amounts in animal tissues where they function as lipid-soluble antioxidants which protect the integrity of cellular and subcellular membranes against oxidative damage. The antioxidant capacity of vitamin E is due to its orientation in biological membranes and its ability to quench free radical reactions by donating a phenolic hydrogen to the radical species. Epidemiological evidence points to an important role for antioxidant nutrients in reducing morbidity and mortality from many chronic diseases including coronary heart disease. Gey et al. 1 observed a strong inverse correlation (r2= 0.63, p= 0.002) between plasma vitamin E and mortality from ischemic heart disease in a cross-cultural study involving 16 European populations. Oxidation of LDL is now considered to be a crucial step in the pathogenesis and progression of atherosclerosis. LDL is protected against oxidation by its endogenous antioxidantsincluding vitamin E, B-carotene, lycopene and others. The maintenance of sufficiently high vitamin E concentrations in LDL may be critical in the protection of LDL against oxidation. Recent studies have demonstrated that vitamin E supplementation increases plasma and LDL vitamin E concentrations and results in a decreased susceptibility of LDL to oxidation in vitro 2'3. These findings suggest that vitamin E and other micronutrients with antioxidant properties could play a major role in future strategies for atherosclerosis prevention. References
1. Gey, K. F., Puska, P., Jordan, P. & Moser, U. K. (1991). Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am. J. Clin. Nutrition 53: 326S334S. 2. Dieber-Roteneder,M., Puhl, H., Waeg, G., Striegl, G. & Esterbauer, H. (1991). Effect of oral supplementation with D-ct-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J. Lipid Research 32: 1325-1332. 3. Jialal, I. & Grundy, S. M. (1992). Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein. J. Lipid Research 33: 899-906.
CORONARY THROMBOLYSIS CAUSES INCREASED EXCRETION OF 8-EPI-PROSTAGLANDIN F2a: NONINVASIVE EVIDENCE FOR FREE-RADICAL PRODUCTION FOLLOWING REPERFUSION. N. Delanty, N. Murphy, J. A. Lawson, G. A. FitzGerald, D. J. Fitzgerald. Centre for Cardiovascular Science, University College Dublin, and Mater Hospital, 41 Eccles Street, Dublin 7. Considerable evidence exists suggesting that the generation of free-radicals following coronary thrombolysis and reperfusion is important in the pathogenesis ofreperfusion injury and myocardial stunning(1). 8-Epi-prostaglandin(PG) F2~is a free-radical derived product of arachadonic acid, with potent biological properties, including platelet activation and vasoconstriction, effects that appear to be mediated by a tbromboxane receptor t2). Thus it may be a marker of reperfusion injury, and may play a role in the increased platelet activity and impaired coronary blood flow that occurs following thrombolysis. We measured the urinary excre-
105
tion of 8-epi-PGF2~ by gas chromatography-mass spectrometry in a canine model of coronary thromholysis. Urinary 8-epiPGF2~ (mean_+SEM) was unaltered following coronary thrombosis and occlusion (196__54 versus 176__39 pg/ktmol creatinine at baseline, n=3). However, in all cases excretion was increased (270-Z-_36pg#tmol creatinine) following thombolysis and reperfusion induced with tissue-type plasminogen activator. Furthermore, in a patient with clinical and angiographic evidence of reperfusion who was treated with streptokinase following an acute anterior myocardial infarction, urinary 8-epi-PGF2~ levels increased from 23 pg#tmol creatininebefore thrombolysis to 84 pg#tmol creatinine after thrombolysis. Levels gradually declined towards baseline over 3 days. These data provide non-invasive evidence of freeradical generation following coronary thrombolysis and reperfusion. 8-Epi-PGF2~ may play a role in the pathogenesis of reperfusion injury. References
1. Loesser, K. E., Kukreja, R. C., Kazziha, S. Y., Jesse, R. L., Hess, M. L. Oxidative damage to the myocardium: a fundamental mechanism of myocardial injury. Cardioscienee 1991; Vol. 2 (4): 199-216. 2. Takahashi, K., Nammour,T. M., Fukunaga, M., Ebert, J., Morrow, J. D., Roberts, II, L. J., Hoover, R. L., Badr, K. F. Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F~, in the rat. Evidence for interaction with thromboxane A. receptors. J. Clin Invest. 1992; 90: 136-141.
SMOKING IS ASSOCIATED WITH INCREASED LEVELS OF A FREE-RADICAL DERIVED PROSTAGLANDIN, 8EPI-PROSTAGLANDIN F2. N. Delanty, J. A. Lawson, D. J. Fitzgerald, G. A. FitzGerald. Centre for Cardiovascular Science, University College Dublin, and Mater Hospital, 41 Eccles Street, Dublin 7. 8-Epi-Prostaglandin(PG)F2~ is a recently discovered prostaglandinwhich is formed as a result of free-radical peroxidation of arachidonic acid "). It has marked biological activity, causing platelet activation and vasoconstriction, effects that are mediated by a thromboxane receptor ~2). It may play a role in the pathophysiology of diseases which are associated with the formation of oxygen radicals. Production of this prostaglandin may also be a marker of free-radical injury. For example, in a patient who had taken a significant paracetamol overdose, a situation which is associated with oxidative stress, urinary 8-epi-PGF2Q levels were 378 pg/I.tmol creatinine (normal range: 5-25 pg/I.tmol creatinine). We measured (by gas chromatography-mass spectrometry) urinary 8-epi-PGF~,~ in 4 young healthy smokers of greater than 15 cigarettes per day and 4 young healthy nonsmokers. Smokers had a greater than 3 fold increase, with a mean (+SD) excretion of 48+8.2 pg/p.mol creatinine in smokers versus 14.5+7.7 in non-smokers (p<0.01). Thus, free-radical injury occurs in healthy subjects who smoke. The formation of the metabolite, 8-epi-PGF2~, may act as one of the mediators of accelerated atherogenesis in smokers. Because 8-epi-PGF2,~ is formed by a cyclooxygenase-independent mechanism, its production would be expected to be unaffected by aspirin therapy. References:
1. Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, K. F., Roberts 11, L. J. A series of prostaglandin F~-like compounds are produced in vivo in humans by a non-cycl6oxygenase, free radicalcatalysedmechanism.Proc. Natl. Acad. Sci. U.S.A. 1990;87:93839387. 2. Morrow, J. D., Minton, L. J., and Roberts 1I, L. J. The F2 isoprostane, 8-epi-prostaglandin F2~,, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/ endoperoxide receptor antagonist. Prostaglandins 1992; 44: 155-163.
106 Royal Academy of Medicine in Ireland" MORPHOLOGICAL CHARACTERISTICS OF OXIDANTINDUCED DAMAGE TO THE VASCULAR ENDOTHELIUM. E. M. Smyth, D. McCole, C. O'Connell and A. K. Keenan. Department of Pharmacology, University College, Belfield, Dublin 4, Ireland. Excess production of reactive oxygen derived free radicals and an abnormal endothetium are associated with many disease states such as atherosclerosis and acute hypertension<1). In this study we examined the morphological alterations that occur during exposure of the endothelial monolayer to H202. Bovine pulmonary artery endothelial cells (BPAEC) were maintained and subcultured as previously described ~2~. Cells grown on 25 mm filters or 10 mm glass coverslips were exposed to H~O2 for 1 hr at 37~ and the resulting cellular damage was assessed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Cell viability was quantified by measuring cellular uptake of neutral red in cells treated under these conditions. TEM of cells treated with H202 showed extensive damage to the mitochondria, plasma membrane and cellular junctions. In addition there appeared to be some blebbing of cellular contents. In SEM apparent cellular retraction was evident leading to the appearance of gaps in the monolayer. H202 dose dependently reduced cell viability as shown in Table I. Table I. Effect of H202 on cell viability [H~O2] % viability 0 100 lmM 53.03 + 8.05 10 mM 22.48 -/- 12.08 100 mM 4.25 + 1.22 In summary, H202 produced extensive morphological damage to the endothelial monolayer and reduced cell viability. This cellular model may prove useful in the study of oxidant meditated damage. References
1. gubanyi, G. N. Vascular effects of oxygen-derived free radicals. Free radical Biol. Med. 1988: 4, 107-120. 2. Smyth, E., Redmond, E. M., Fenton, M., Keenan, A. K. Neutral endopeptidase does not affect atrial natriuretic factor-stimulated cyclic guaninemonophosphateproductionin bovinepulmonaryarteryendothelial cells. Eur. J. Int. Med. 1992: 2, 285-287.
MK-801, BMY 14802 AND EXIFONE ARE NEUROPROTECTIVE AGAINST ISCHAEMIA-INDUCED BRAIN DAMAGE IN THE GERBIL. M. O'Neill, M. Canney, B. Earley and B. E. Leonard. Pharmacology Department, University College Galway. Cerebral ischaemia caused by either cardiovascular or brain trauma causes a selective pattern of neurodegeneration. The Mongolian gerbil (Meriones ungiculatus) was introduced in 1966 as an animal model of cerebral ischaemia and has been extensively used to test putative neuroprotective agents. The exact mechanism of ischaemic cell death is not fully elucidated, but glutamate is increased after ischaemia. This glutamate acting on N-methyl-D-aspartate (NMDA) receptors initiates a cascade of excitatory amino acid, calcium and free radical mediated toxicity and cell damage. In the present series of experiments the effects of MK-801 (an NMDA antagonist), BMY 14802 (a sigma ligand) and exifone (a free radical scavenger) on locomotor activity 24, 48 and 72 after 5 min bilateral carotid occlusion (BCO) and on histological parameters 96 hours after surgery was exainined. Sham operated
LJ.M.S. February, 1994 animals were used as controls (the animals were anaesthetised with 2% halothane delivered with 02 at 1 litre/min via a face mask). The drugs were administered 1 hour before 5 rain BCO at the following doses: MK-801 (2.5 mg/kg i.p.); BMY 14802 (1 and 10 mg/kg i.p.) and exifone (500 and 1000 mg/kg p.o.). Results indicated that 5 min BCO caused a large increase in locomotor activity 24, 48 and 72 after surgery. Histological investigation indicated that the CA1 region of the hippocampus was extensively damaged in 5 min BCO animals. MK-801 attenuated the hyperactivity 72 hours after surgery and was neuroprotective as shown by histological methods. BMY 14802 and exifone did not reduce the locomotor activity, but were neuroprotective. These results show that drugs acting on the NMDA receptor and free radical scavenging drugs have neuroprotective effects against ischaemia-induced brain damage in the gerbil.
FEEDING FISH OIL COMPARED WITH CORN OIL DOES NOT INCREASE DNA DAMAGE IN HEART AND AORTA CELLS OF HAMSTERS FED COPPER AND VITAMIN E DEFICENT DIETS. E. Turley, V. J. McKelvey-Martin, J. J. Strain. Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA. Concurrent with the recognition of dietary fish and fish oils as therapeutic agents in coronary heart disease, concern has been raised that increased polyunsaturated fatty acid ingestion leads to increased oxidative stress. Vitamin E is the major lipid soluble chain breaking antioxidant while copper is essential for the copper dependent antioxidant enzyme superoxide dismutase. We investigated the influence of fish oil (15% w/w) in diets containing either adequate or deficient levels of both vitamin E and copper on DNA strand breakage as assessed by the comet assay. A total of 48 Male Syrian hamsters were divided into 6 groups (n=8) and fed the various diets for 14 weeks. The results for each group (mean + S.E.) are shown in the table. Dietary group Tail moments Physical area Heart Aorta H e a r t Aorta Corn oil+Vitamin E+Copper 18.3+.1 18.4:1:2.8 1343+210 1427+157 Corn oil+Vitamin E 24.0+_.2.4 15.8+3.0 1581+155 1474+184 Fishoil+VitaminE+Copper 21.3+4.2 25.4+4.6 1324+165 1502.-1:128 Fishoil+VitaminE 23.5+3.3 13.3+3.0 1446+151 1122+100 Fishoil+Copper 22.6+4.1 25.4+4.6 1449-2_125 1313+124 Fish oil 22.0-J:1.7 20.5+2.7 1308+143 15705:187 Two-way ANOVA indicated that there was no significant difference between groups. Fish oil ingestion at this relatively high level both in the presence and absence of dietary copper and vitamin E does not appear to increase DNA damage in hamsters.
LOW INTENSITY LASER IRRADIATION (LILI), AT THERAPEUTIC DOSES, INDUCES SUPEROXIDE ANION (02-) PRODUCTION IN THE MACROPHAGE-LIKE CELL LINE U937 G. A. Gallagher, T. D. Shields, S. O'Kane, S. J. Eason, B. M. Hannigan, W. S. Gilmore, J. M. Allen. Biomedical Sciences Research Centre (Departments of Occupational Therapy & Physiology and Biological & Biomedical Sciences), University of Ulster at Jordanstown, BT37 0QB, N. Ireland. Low Intensity Laser Irradiation (LILI) may alter the proliX'-
Vol. 163
Section of Biomedical Sciences 107
No. 2
eration of cells associated with wound repair. Macrophages are tissue cells which secrete mediators of wound repair so it is probable that laser biostimulation influences macrophage functiont~). This study determined the effect of LILI (660nm, 15mW, 5kHz) on superoxide anion (02-) production in the macrophagelike cell line U937, both undifferentiated and following differentiation with dimethyl sulphoxide. Superoxide production was based on the reduction of ferricytochrome C. Cells were exposed to laser at energy densities between 1 and 11.5 J/cm 2and to PMA, a known potent inducer of 02-. Negative controls included nonirradiated cells. Superoxide production was measured over a 90 minute period. Preliminary results indicate that differentiated U937 cells respond to PMA by producing 02-. These cells also showed a dose dependent response to laser irradiation. Superoxide was produced at energy densities of 1.0 and 2.9 J/cm 2, although with a time-course different to that for PMA. Reactive oxygen species (ROS) such as 02- are known to be intracellular messengers for the activation of transcription factors e.g. NF-KB and AP-1 <2). We may now hypothesise that a molecular basis for laser biostimulation is the alteration of gene expression through production of intracellularROS. Other forms of non-ionising radiation e.g. UVC are thought to work in a similar manner. References
1. Young, S., Bolton, P., Dyson, M., Harvey,W. and Diamantopoulos C. Macrophage responsiveness to light therapy, Lasers. Surg. Med. 1989: 9; 497-505. 2. Schreck, R., Rueber, P., Baeuerle, P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of NFKB transcription factor and HIV-1, EMBO. J. 1991: 10(8); 2247-2258.
ensure DNA integrity and/or (b) directly related to oxygen metabolism, is being investigated.
IN VIVO ACTIVATION OF A NOVEL BIOREDUCTIVE ANTI-TUMOUR AGENT. M. Hejmadi ~, S. R. McKeown 1, J. J. A. McAleer 2, L. H. Patterson 3. ~Department of Biological and Biomedical Science, University of Ulster, Jordanstown, N.lreland. 2Departments of Haematology & Oncology, Queen's University, N. Ireland. 3Department of Pharmacology, De Montfort University, Leicester, U.K. It has been reported previously that N-oxides of some drugs may be bioreductively activated to more cytotoxic molecules. We have investigated a group of alkylaminoanthraquinones which become DNA affinic when reduced. The in vivo cytotoxicity ofAQ4N (1,4-bis- {[2-(dimethylamino-N-oxide)ethyl]amino} 5,8 dihydroxy anthracene-9,10-dione) was assessed in the T50/80 mammary carcinoma grown on the rear dorsum of male BDF mice. When the tumour reached 6.5 mm diameter a range of drug doses was administered with or without hypobaric hypoxia. Drug effects were assessed as the time to double treatment volume. A dose enhancement ratio (DER) was determined by comparing the dose of drug required to give an equivalent antitumour effect in air as compared to hypobaric hypoxia. AQ4N showed a significantly greater anti-tumour effect in hypobaric hypoxia than in air (ANOVA, p = 0.02) with a DER approaching that of SR 4233 ~l). A disadvantage of SR 4233 was the marked dose dependent toxicity, measured by weight loss, an effect that was significantly enhanced by hypobaric hypoxia. In comparison AQ4N showed only marginal toxicity even with high drug doses and hypoxia. Reference
OXIDANT INDUCED BIOMOLECULE DAMAGE AND THE AGEING PROCESS C. M. King, Y. A. Wilkinson and P. G. McKenna. Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, N. Ireland BT52 1SA. Endogenously generated oxidants, produced as a consequence of normal metabolism, can cause biomolecule damage. Imperfections in defence systems which maintain DNA integrity may lead to accumulating mutations which, on their own or in combination with other age-related changes, may contribute to ageing and development of age-related pathologies. In this ethically approved investigation, mutation frequency at the hypoxanthine guanine phosphoribosyl transferase locus was assessed in lymphocytes isolated from male volunteers (age groups 35-39 and 65-69 years). Results showed that the mutation frequency in the 65-69 years group was approximately twice that in the the 35-39 years group (2.49/106 cells and 1.04ll06cells respectively). An increased frequency of chromosomal aberrations was evident in the 65-69 years group. Blood levels of glutathione peroxidase, superoxide dismutase, catalase, caeruloplasmin, uric acid and bilirubin were also measured. A significant decrease in mean catalase activity with age from 58 sec-~/gHb in the 3539 years group to 39 sec-1/gHb in the 65-69 years group was observed. No significant changes occurred in the levels of the other antioxidants. That the observed increase in mutations with age is (a) due to a decreased efficacy of the mechanisms which
1. McAleer, J. J. A., McKeown, S. R., MacManus, M. P., Lappin, T. R. J., Bridges, J. M. Hypobaric hypoxia: a method for testing bioreductive drugs in vivo. Int. J. Radiat. Oncoi. Biol. Phys. 1992; 23: 551-555.
THE ANTI-TUMOUR EFFECT OF BIOREDUCTIVE DRUGS: GENDER EFFECTS. S. R. McKeown 1, J. J. A. McAleer 2, P. S. C. Gray 3, T. R. J. Lappin3, J. M. Bridges 3. 1Department of Biological and Biomedical Sciences, University of Ulster, Jordanstown, NexCtownabbey BT37 OQB, Departments of 2Oncology and 3Haematology, The Queen's University of Belfast, N. Ireland. Hypobaric hypoxia has been used to induce tumour hypoxia for in vivo comparison of the anti-tumour effects of bioreductive agents "). BDF mice bearing the T50/80 mammary carcinoma were treated with the bioreductive agents SR 4233 and RSU 1069 over a range of doses under normobaric (oxic) and hypobaric (hypoxic) conditions. The time taken for the tumour to double treatment volume (VDT) was used as a measure of anti-tumour effect. Analysis of the results allowed dose enhancement ratios (DER) to be determined for oxic and hypoxic conditions. The DERs for SR 4233 and RSU 1069 in male mice were 8.8 and 8.5 respectively, showing these agents had an equivalent
108 Royal Academy of Medicine in Ireland and substantial enhancement of their cytotoxicity when combined with hypobaric hypoxia. For female mice these agents caused considerably less growth delay and hypobaric hypoxia caused minimal enhancement of their anti-tumour effects. For SR 4233 weight loss following treatment correlated closely with drug dose. Thus this drug showed similar systemic toxicity in male and female hosts, although the anti-turnout effects were widely different. Tracer doses of the hypoxic cell labelling agent 3H-Misonidazole were used to analyse the extent of hypoxia in a range of tissues for both sexes. Reference
1. McAleer, J. J. A., McKeown, S. R., MacManus, M. P., Lappin, T. R. J., Bridges, J. M. Hypobaric hypoxia: a method for testing bioreductive drugs in vivo. Int. J. Radiat. Oncol. Biol. Phys. 1992; 23: 551-555.
ANTIOXIDANT ENZYMES AND DNA REPAIR: BOTH PREVENT CELLULAR ACCUMULATION OF ROSINDUCED DNA DAMAGE B. M. Hannigan, S.-A. M. Richardson, Y. A. Wilkinson, P. G. McKenna. Department of Biological and Biomedical Sciences, University of Ulster at Coleraine, N. Ireland. The apparent relationship between ROS and human disease may be studied in vitro using ROS at concentrationS in excess of those which mediate normal physiological processes. Two murine cell lines (707 Friend erythroleukaemia (FEL) and L5178Y lymphoma) were used to evaluate the roles of antioxidant enzymes and DNA excision repair in preventing the accumulation of ROS mediated DNA damage. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured in each cell type. The activities of all three were greatest in FEL cells. The greatest difference was in CAT levels, Both CAT and GPx scavenge H202but CAT is active only with raised H202 levels. Thus L5178Y may be deficient in their ability to scavenge H20 2. Xanthine (1.5mM) + xanthine oxidase (1.5mU/ml) (x+xo) was the source of ROS, both 0 2- and H202. Damage induced by x+xo was estimated using cytogenetic end-points including cell survival and point mutation frequency (pt. mut.) at the HGPRT locus. DNA excision repair was estimated as unscheduled DNA synthesis.
I.J.M.S.
February, 1994 FEL LS178Y Pre-ROS Post-ROS Pre-RoS Post-ROS *Survival(%) 100 91.00"&-0.01 I00 38.80-2-0.01 *pt. mut. (xl0-6) 2.03_+0.1 2.95:L-0.02 9.01+0.01 36.00-Z-~.02 *DNA synthesis(% cells) 72.30"2_4.7 59.805_-6.2 71.g0~:1.3 62.70-20.2 *DNArepair(% cells) 21.80-&-_1.3 37.85+9.8 16.40+_.2.7 21.10-3:1.2 *Mean+ s.d, n = three separate experiments In conclusion, ceils with insufficient antioxidant defences may be susceptible to DNA damage, those deficient in DNA excision repair (e.g. L5178Y) subsequently dying or expressing mutations. This work was supported by DHSS (N.I) and by the Ulster Cancer Foundation. S.-A. M. R was a D.E.N.I. Distinction award student.
THE EFFECTS OF DICLOFENAC SODIUM ON THE HEALING OF FREE GRAFTED SKELETAL MUSCLE P. G. Murphy, N. Davidson, A. C. B. Hooper. Department of Human Anatomy and Physiology, University College, Earlsfort Terrace, Dublin 2. Bilateral free grafting of the extensor digitorum longus muscle was carried out in adult male Sprague-Dawley rats under general anaesthesia (Pentobarbitone sodium 5 mgs/kg body weight i.p.). Treated animals received one daily intramuscular injection of diclofenac sodium (Voltarol Ciba Geigy 5 mgs/kg body weight). Transverse sections of the muscles were examined by light microscopy at days 5 and 10 postoperatively and compared with untreated controls (N > 15 muscles). On day 5 the proportionate area of inflammationof the treated muscles, 25 + 3%, was significantlyless than that of the controls, 43 -+ 4% (P < 0.01) by Student's t test). After 10 days there were no significant differences between the areas of inflammation and fibre regeneration in treated and untreated muscles (P = 0~94 and 0.45 respectively), nor of their DNA content~, 3.34 _+ 0.14 and 3.19 _+ 0.18 ktg/mg (P = 0.53). Inflammation was reduced, but there was no interference with the process of fibre regeneration. These findings support the use of diclofenac sodium in the treatment of muscle injuries. Reference
1. Giles, K. W., Myeps, A. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 1965: 206, 93.