Cent Of Control Values Research Articles

Effect of postconditioning on mitochondrial dysfunction in experimental aortic cross-clamping

Cross-clamping of the aorta during abdominal aortic aneurysm surgery induces muscle ischaemia with resultant morbidity. This study tested whether ischaemic postconditioning would decrease mitochondrial dysfunction in skeletal muscle by reducing oxidative stress. Three groups (9 rats each) underwent surgery, including a control group without ischaemia and an ischaemia-reperfusion group that had 3 h ischaemia induced by aortic clamping and collateral vessel ligation, followed by 2 h of reperfusion. The third group had ischaemia for 3 h then underwent postconditioning comprising three short intervals of ischaemia-reperfusion at the onset of reperfusion. Activity of complexes I, II, III and IV of the mitochondrial respiratory chain was monitored in gastrocnemius muscle, along with oxidative stress measured by dihydroethidium (DHE) staining and antioxidant defence determined by measurement of glutathione levels. Ischaemia-reperfusion alone caused a significant reduction in maximal oxidative capacity (-31.8 per cent; P = 0.002), activity of complexes II, III and IV (-34.5 per cent; P = 0.007) and complex IV activity (-30.6 per cent; P = 0.039). It also increased reactive oxygen species (DHE staining increased to 223.1 per cent of control value; P = 0.027) and reduced antioxidant defence (glutathione level -28.6 per cent; P = 0.039). Postconditioning counteracted these deleterious effects by increasing mitochondrial complex I, II, III and IV activities, restoring muscle DHE staining and preserving glutathione content. Ischaemic postconditioning protects skeletal muscle mitochondria against ischaemia-reperfusion injury by reducing oxidative stress and preserving antioxidant defence in an experimental model. Mitochondrial protection to reduce reperfusion injury in clinical vascular surgery may be warranted.

Differences in contractile properties of anorectal smooth muscle and the effects of calcium channel blockade.

Pharmacological manipulation of the anal sphincter is hampered by a lack of specificity. This study aimed to determine differences in the role of intracellular and extracellular calcium in the development of tone and agonist-induced contractions between internal anal sphincter (IAS) and rectal circular muscle which might allow targeted manipulation. Smooth muscle strips from the IAS and rectal circular muscle of 24 Large White pigs were mounted for isometric tension recording in a superfusion organ bath in the presence of different perfusates. IAS developed tone and spontaneous activity that were abolished by nifedipine, which also reduced contractions to noradrenaline to 72 per cent of control values. Rectal smooth muscle developed spontaneous activity but no tone. Nifedipine abolished the activity and reduced contractions to carbachol to 17 per cent of control. Contractile activity was abolished in both tissues in calcium-free solution. Transient exposure to a high calcium concentration reloaded the stores, and the ability of agonists to release stored calcium was tested after 3 min in calcium-free solution. In IAS, noradrenaline contraction was 76 per cent of control and in rectal circular muscle carbachol contraction was 57 per cent of control. Store loading was prevented by nifedipine in rectal smooth muscle but not IAS. Cyclopiazonic acid reduced store filling in both tissues. Agonist-induced contraction of IAS is largely due to release of stored calcium and L-type calcium channels are not needed for store filling. Rectal circular smooth muscle depends more on extracellular calcium and uses L-type calcium channels for agonist-induced contraction and store filling. These differences suggest that targeted manipulation may be possible in patients with anorectal disorders.

Effect of transforming growth factor-β on the plasminogen activator system in cultured first trimester human cytotrophoblasts

Plasminogen activators (PAs) play an important role in facilitating trophoblast invasion of the uterus and in the maintenance of blood fluidity within the placental intervillous spaces. We previously found that transforming growth factor-β (TGF-β), produced mainly by the decidua, inhibits first trimester trophoblast invasiveness at least partly via induction of tissue inhibitor of metalloproteinases-1 production and secretion by the trophoblasts. The present study examined whether TGF- β 1 affects PA and plasminogen activator inhibitor-1 (PAI-1 ) production by cultured human cytotrophoblasts. Immortalized first trimester human cytotrophoblasts (HTR-8/SVneo) were cultured in the absence or presence of TGF- β 1 (0–10 ng/ml) for 24 h, after which the levels of urokinase-type PA (uPA), PAI-1 and uPA activity in the serum-free conditioned media were determined by enzyme-linked immunosorbent assay (ELISA), protein zymography, and a chromogenic uPA activity assay. Cellular PAI-1 mRNA levels were also determined in cultures following a 24-h incubation with a single dose (5 ng/ml) of TGF- β 1. Presence of TGF- β 1 at 1 ng/ml resulted in a greater than 12-fold reduction in the levels of total uPA as determined by ELISA. Furthermore, released uPA activity levels in similar cultures incubated with 5 ng/ml of TGF- β 1 were reduced to 35 per cent of control values. In contrast, cultures incubated with as little as 0.1 ng/ml TGF-β exhibited a 63 per cent increase in the levels of secreted PAI-1 protein. Similarly, both the 2.2- and 3.0-kb PAI-1 cellular mRNA species were elevated in trophoblast cells incubated with 5 ng/ml TGF- β 1 when compared with control cells. Thus, it appears that the reduced uPA activity observed in the cultures incubated with TGF- β 1 is due to reduced secretion of uPA and increased PAI-1 production and secretion. These results suggest that TGF-β may also exert its anti-invasive effect by down-regulating trophoblast-derived PA activity. Through its effects on the PA system, TGF-β may also play an indirect role in the regulation of uteroplacental blood flow.

Fasciola hepatica infection in sheep: changes in liver metabolism

Several aspects of liver function during infection with Fasciola hepatica were examined in sheep four weeks after infection and compared with the changes observed in infected rats. Previously reported respiratory abnormalities in mitochondria isolated from the left lobe of the liver of infected sheep were characterised further. Evidence is presented that the respiratory lesion is located in the mitochondrial electron transport chain and that the aberrant respiratory behaviour is not associated with an increase in non-esterified fatty acids and the depletion of mitochondrial phospholipids, as is the case in the rat. Microsomal membranes, which have also been shown to be depleted of phospholipids in the fluke-infected rat liver, showed no such changes in the sheep. However, in common with the rat, a substantial loss of cytochrome P 450 was recorded in microsomes prepared from the left lobe, and the glycogen content of the left lobe was found to be less than 50 per cent of control values. No change was observed in glucose 6-phosphatase activity. All these changes were localised effects, confined to areas of fluke infiltration.

Molecular prenatal diagnosis of 3-hydroxy-3-methylglutaryl CoA lyase deficiency.

We report the first molecular prenatal diagnosis of 3-hydroxy-3-methylglutaryl CoA lyase (HL) deficiency. The proband had a classic but severe presentation with hypoketotic hypoglycaemia and acidosis, secondary mental retardation, and epilepsy, and HL deficiency was documented in cultured fibroblasts. We found him to be homozygous for the frameshift mutation N46fs (+1), which yields a distinct pattern on single-strand conformation polymorphism (SSCP) analysis. In two subsequent pregnancies, molecular prenatal diagnosis was performed using SSCP. In the first, chorionic villus biopsy was normal. In the second pregnancy, amniocentesis revealed an affected fetus. In both pregnancies, the diagnosis was confirmed enzymatically. HL activity was less than 7 per cent of control values in amniocytes and fetal liver of the affected pregnancy. In the second pregnancy, amniotic fluid metabolite measurements by stable isotope dilution-selected ion monitoring mass spectrometry showed greater than 100-fold increases of 3-hydroxy-3-methylglutaric acid and of 3-methylglutaconic acid levels compared with controls.

Comparative Cytotoxic Effects of Short-chain Fatty Acids Produced by Periodontal Pathogens on Two Cultured Fibroblast Lines

The cytotoxic effects of a range of short-chain fatty acids produced by anaerobic periodontopathic bacteria were compared using two types of fibroblast culture: Vero cells and human gingival fibroblasts. Morphological changes in cells exposed to 1.56—25 mM acetic, n-butyric, isobutyric, n-caproic, isocaproic, phenylacetic, propionic, n-valeric and isovaleric acids for three days were monitored using phase contrast microscopy and metabolic activity of cultures assessed by measuring reduction of the tetrazolium salt MTT. Vero cells were considerably more sensitive than human gingival fibroblasts to both morphological and metabolic changes. Reduction in metabolic activity of Vero cells was n-butyric > propionic, n-valeric and isovaleric > phenylacetic, n-caproic and isocaproic. N-butyric acid (≥ 12.5 mM) caused progressive cell rounding and death of >90 per cent of the cells by three days. Exposure to n-caproic, n-valeric and isovaleric acids (25 mM) produced spindle-shaped Vero cells with one or more long dendritic projections and reduced metabolic activity to ≤60 per cent of control values ( P <0.001). Comparable morphological changes were not observed in human gingival fibroblasts exposed to the maximum concentrations of any acid and only n-butyric, propionic and phenylacetic acids reduced metabolic activity significantly. Thus although Vero cells have traditionally been used to test for the cytotoxic effects of potential virulence factors from periodontal pathogens, their behaviour may not be representative of the cells normally exposed to these components in vivo . Keywords - Cytotoxicity, Short-chain fatty acids, Human gingival fibroblasts, MTT, Vero cells.

Effect of aging on myo-inositol and phosphoinositide metabolism in the cochlear and vestibular sensory epithelia of the rat

Neurotransmission and transmembrane signaling are among the cellular mechanisms affected in the aging nervous system. In the inner ear, the phosphoinositide second messenger cascade is of particular interest as a target of the aging process. In both the cochlear (CSE) and vestibular sensory epithelia (VSE), the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP 2) to the second messenger inositol 1,4,5-trisphosphate (InsP 3) is coupled to muscarinic cholinergic and P 2y purinergic receptors and may be linked to calcium homeostasis. The present study compared the turnover of phosphoinositides (PtdlnsPs), receptor-mediated release of inositol phosphates (InsPs), and concentrations of endogenous myo-inositol in the CSE and VSE of young (3 months) and aged (24 months) Fischer-344 rats. In the aged rat, there was a significant increase in [ 3H]inositol incorporation (per mass of protein) into PtdlnsPs plus InsPs in both sensory epithelia while the protein content remained unchanged. In contrast, no age-dependent differences were found when pre-labeled [ 3H]PtdInsPs were ‘chased’ with non-radiolabeled myo-inositol indicating that the turnover of these lipids was unaffected. The cholinergic receptor agonist carbamylcholine and the P 2 purinergic receptor agonist adenosine 5′- O-(3-thiotriphosphate) stimulated the release of [ 3H]InsPs two- to six-fold in both organs. This agonist-stimulated release of [ 3H]InsPs (per mass of protein) was significantly higher in aged animals. However, when the same stimulation was expressed as per cent of control values, there was no age-dependent difference. Finally, the concentration of endogenous myo-inositol decreased by 44% in the aged CSE and by 24% in the aged VSE. In contrast, levels of added myo-[ 3H]inositol were higher in aged tissues. These results suggest that the increased labeling of PtdlnsPs and InsPs in the aged CSE and VSE is a consequence of the increased specific radioactivity of the myo- [ 3H]inositol precursor pool. The activity of the phosphoinositide second messenger pathway thus appears unchanged. However, a decreased myo-inositol content may contribute to age-dependent pathology in these tissues, myo-inositol is an organic osmolyte and volume regulator. Changes in osmotic pressure or turgor of hair cells could alter micromechanical coupling on the basilar membrane and vestibular epithelium causing pathophysiological changes in sensory transduction.

Different growth responses to agents which elevate cAMP in human melanoma cell lines of high and low experimental metastatic capacity

Human melanoma variants of low and high experimental metastatic activity, which had been derived from the same parental line, showed markedly different growth responses to agents which elevated intracellular cAMP. The high metastatic line had a significant decrease in in vitro proliferation following treatment with cholera toxin (10(-9) M) and forskolin (100 microM), with both agents causing virtual cessation of cell growth after 3-5 days incubation. Pre-treatment with 10(-9) M cholera toxin reduced colony forming ability to 11-15 per cent of control values, saturation densities were decreased to 10-25 per cent of controls and these cytostatic responses were accompanied by changes in cellular morphology. Lung colonising capacity of this cell line after i.v. injection into athymic mice was reduced significantly by prior exposure to cholera toxin (a median of 2 lung nodules versus 26 lung nodules for untreated, control cells). In contrast, low metastatic cell lines showed no significant growth inhibition in the presence of these agents. Cholera toxin (10(-9) M) reduced colony forming ability of these cells to only 74 per cent of control values and there were no significant decreases in growth rate nor any morphological changes in response to either cholera toxin or forskolin. The variable response obtained in the cell lines appeared neither to be a consequence of variation in induced levels of intracellular cAMP nor in differences between the cell lines in response to the same agent; forskolin (100 microM) induced a maximal 25-fold elevation and cholera toxin (10(-9) M) a 2.5-fold elevation increase in cAMP. These data show that highly metastatic variants of a human melanoma cell line differ from their less metastatic counterparts in the way they respond to agents which elevate the second messenger molecule cAMP.

Mannosidosis in a litter of Persian cats.

Three kittens in a litter of Persian cats showed, from the age of eight weeks, tremor, ataxia, dysmetria, progressive weakness and emaciation. Cytoplasmic vacuolation was observed in neurons, mesenchymal and epithelial cells of tissues taken post mortem. The alpha-mannosidase activity of brain tissue of one cat tested was 4.8 per cent of control values and the urine of two cats contained large amounts of mannose-rich oligosaccharides.

Effect of cutting or crushing the rat sciatic nerve on synthesis of substance P by isolated L5 dorsal root ganglia

Following cutting or crushing the rat sciatic nerve, synthesis of substance P by L5 dorsal root ganglia in vitro was reduced respectively to 20 and 40 per cent of control values. By day 64 the ability to synthesise substance P had been fully restored in crushed neurones but in cut neurones remained at a low level. We conclude that substance P synthesis is a more sensitive index of the effect of nerve injury than is substance P content and further that regenerating axons are able to support substance P synthesis before they reach their target tissue.

36Cl − and 86Rb + uptake in rat parotid acinar cells

36Cl − uptake was markedly (85 per cent) inhibited by the loop diuretics, furosemide and bumetanide. Partial replacement of Na + in the incubation medium (from 137 to 50 mM) reduced 36Cl − uptake 30 per cent; total replacement reduced uptake to 40–45 per cent of control values. Partial replacement of K + (from 5.8 to 1 mM) decreased 36Cl − uptake approx. 45 per cent; complete replacement resulted in minimal levels of 36Cl − uptake (< 10 per cent of controls). Ouabain reduced 36Cl − uptake approx. 55 per cent in the absence of bumetanide, but was without effect in its presence. 86Rb + uptake was reduced approx. 85 per cent with bumetanide; complete replacement of Cl − with I − or gluconate −, decreased 86Rb + uptake by 55 or 40 per cent respectively. The results support the notion that the bulk of Cl − influx in rat parotid acinar cells is via a loop diuretic-sensitive Na +/K +/Cl − co-transport mechanism as may occur in other secretory epithelia.

Effect of tricyclohexylhydroxytin on synaptosomal Ca2+-dependent ATP hydrolysis and rat brain subcellular calmodulin.

Effect of tricyclohexylhydroxytin (plictran) on Ca2+-ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+-ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+-ATPase in a concentration-dependent manner. A complete reversal of calmodulin activation of Ca2+-ATPase was observed with 2-3 nM plictran. A 50 per cent decrease of CaM activated Ca2+-ATPase was observed with 0.5 nM plictran, a concentration at which no significant effect was observed on basal enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+-ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg-1 day-1 treated rats for 3 days respectively. The activity levels of Ca2+-ATPase in 10 and 20 mg plictran kg-1 day-1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.

Isoproterenol-induced changes in glutathione metabolism in the rat parotid gland

Rat parotid glands were removed at timed intervals after intraperitoneal injection of isoproterenol (IPR). Reduced/oxidized glutathione ratios, glutathione peroxidase, glutathione reductase and amylase activities were determined. Parotid gland amylase content fell to less than 5 per cent of control values within l h after IPR, then slowly returned to control levels by 24 h. The ratio of reduced/oxidized glutathione was decreased at 2 h after IPR injection, returned to near control values at 6 h, then increased above control levels at 9, 12 and 24 h. Glutathione peroxidase activity was increased only at 12 h after injection of IPR, whereas glutathione reductase activity initially decreased, then reached peak values at 12h. These results indicate that alterations in metabolic activity during the secretory cycle are accompanied by changes in the reduced/oxidized glutathione ratio and the activity of glutathione-metabolizing enzymes. Cellular events contributing to these alterations in glutathione metabolism may include exocytosis, endocytosis and membrane recycling, increased amino-acid transport and resynthesis of secretory proteins.

Effect of anthelmintic treatment on the productivity of Iambs infected with the intestinal nematode, Trichostrongylus colubriformis

Liveweight gain, food intake, wool growth and concentration of serum constituents were measured in four groups of three-month-old lambs. Groups 1 and 2 were infected on five days each week with 2500 Trichostrongylus colubriformis larvae for 18 weeks. Group 2 lambs were treated with fenbendazole (5 mg kg-1) at week 10 and week 15. Group 3 lambs were similarly infected for 10 weeks, treated with fenbendazole, but given no further larvae. A fourth control group remained uninfected throughout the trial. All lambs were killed at week 20. Mean worm populations were 16,130, 430 and zero for groups 1, 2 and 3 respectively. Over the first 10 weeks of infection, liveweight gain was reduced by 52, 43 and 51 per cent and wool growth by 33, 28 and 27 per cent respectively, in groups 1, 2 and 3. Serum hypophosphataemia and hypoalbuminaemia occurred in all three infected groups. Between weeks 10 and 20 overall weight gain and wool growth in group 1 lambs were 44 and 46 per cent lower than the controls, whereas weight gains of group 2 and group 3 lambs were similar to or slightly higher than in the controls. However, wool growth in these two groups after treatment was only 73 to 74 per cent of control values. Serum phosphorus concentrations increased to control levels within one week of anthelmintic treatment and serum albumin concentrations by two to four weeks. The ability of group 2 lambs to improve their performance after anthelmintic treatment, in the face of continued challenge, was attributed to development of resistance to reinfection.

Amylo-1,6-glucosidase activity in cultured cells: a deficiency in type III glycogenosis with prenatal studies.

Deficiency of amylo-1,6-glucosidase activity was expressed in parallel in liver and skin fibroblasts from a patient with type III glycogenosis. In crude extracts of control liver and muscle, amylo-1,6-glucosidase (M.W. 164000) was identified by immunoprecipitation; no cross-reacting material was found in the patient's liver. Assay of amylo-1,6-glucosidase activity in cultured skin fibroblasts from the affected family revealed less than 10 per cent of control value in mutant homozygous cells whereas in cells from the parents, activity was reduced to 40-60 per cent of the control value. Activity in cultured amniotic fluid cells was similar to that of control fibroblasts. In cultured amniotic fluid cells obtained during the mother's subsequent pregnancy, the normal amylo-1,6-glucosidase activity measured, predicted correctly the outcome of this pregnancy prior to the 20th week of gestation.

Halothane depresses the response of carotid body chemoreceptors to hypoxia and hypercapnia in the cat.

Halothane is known to inhibit the ventilatory responses to hypoxia and hypercapnia. In order to determine whether this inhibition was mediated by peripheral chemoreceptors, the authors measured the effect of halothane on the response of carotid body chemoreceptors to these stimuli. Cats were decerebrated under brief halothane anesthesia, paralyzed, and ventilated. Chemoreceptor activity was recorded from single- or few-fiber preparations of carotid sinus nerve, and the inspiratory drive was recorded from the whole phrenic nerve. Steady-state responses were measured at three levels of CO2 tension (19-92 mmHg) during hyperoxia, and at four levels of O2 tension (35-450 mmHg) at a fixed PaCO2. Both responses were measured before, during, and after 0.5-1.0 per cent halothane was inspired. The halothane inhalation was maintained for at least 30 min before the responses were obtained. Halothane reduced the slope of chemoreceptor response to hypercapnia to about 48 per cent of the control slope. The response to hypoxia was reduced to about 58 pr cent of the control response. The increase in firing after intravenous nicotine (100 micrograms), summed for 20 s, was reduced to 25 per cent of the prehalothane control values; that after NaCN (25 micrograms) was reduced to 17 per cent of the control value. The effect of halothane was prompt (half complete in 1-2 min) and reversible. This finding explains some of the inhibition of the ventilatory responses to hypoxia and hypercapnia caused by halothane.

Glucocorticoids alter the ratio of type III/ type I collagen synthesis by mouse dermal fibroblasts

The type of collagen synthesized by primary cultures of dermal fibroblasts from two different strains of mice (A/J and C57BL6/J) was investigated. Confluent cultures were grown for four days with or without 10 −6 and 10 −7 M corticosterone or 10 −6 M dexamethasone. Glucocorticoid treatment results in a lower type III/type I procollagen ratio in both strains. While the amount of type I procollagen remains unchanged, type III is reduced to about 25 per cent of control values in the cells from both mouse strains. Thus, glucocorticoids can modulate the types of collagen synthesized by cultured cells.

Susceptibility of the Proximal Tubular Brush Border to Acute Obstructive Injury

The purpose of this investigation was to determine the susceptibility of proximal renal tubular epithelial cells to the effects of acute urinary tract obstruction. Brush border derived-renal tubular epithelial antigens (RTE) were quantitated in urine by radioimmunoassay both before and after the induction of bilateral ureteral obstruction of 15-150 minutes duration in anesthetized diuretic rats. Glomerular filtration rates (GFR) before and after obstruction were determined by calculating renal clearances of Na iothalamate 125I. Light microscopic examination of renal tissue was performed.Post obstructive renal function was well preserved in all rats with GFRs ranging from 76–100 per cent of control values. However, in the immediate post-obstructive period RTE excretion increased dramatically in all rats. Up to 10 fold increases in urine antigen concentrations were observed under conditions of isosthenuria and stable urine flow rates. The degree of elevation correlated with the length of obstruction and with the severity of histologic changes in the proximal tubules (progressive proximal tubular dilatation and brush border loss). It is concluded that proximal tubular cells are highly susceptible to obstructive insults as brief as 15 minutes in duration. The mechanism of this injury and its effect on post-obstructive renal function remain to be clarified.

Relationship of brain cyclic nucleotide levels and the interaction of ethanol with chlordiazepoxide

The effects of combined administration of ethanol (4 g/kg) and chlordiazepoxide (CDP, 12.5 mg/kg) on mouse brain c-AMP and c-GMP levels were investigated in order to test the hypothesis that the supra-additive effect of CDP on ethanol sleep time may be related to a supra-additive alteration in brain cyclic nucleotide levels induced by the combined drugs. Ethanol alone or CDP by itself did not cause any change in brain c-AMP levels, except for a transient decrease in the cerebral cortex and midbrain at 0.5 hr after ethanol injection, as well as a transient increase in the cerebellum at 0.5 hr after CDP injection. The combined drug treatment did not result in a supra-additive effect on c-AMP levels. On the other hand, c-GMP levels were depressed significantly for 4 hr after ethanol injection, especially in the cerebellum. The mice regained the righting reflex when the c-GMP levels were still about 30 per cent of control values. Ethanol and CDP together induced a supra-additive decrease of c-GMP concentrations in the cerebellum at 2 and 4hr. This resulted in a lengthened period (about 2.5 hr) during which the cerebellar c-GMP levels were below 30 per cent of control values, and this interval coincided with the increase in sleep time, suggesting a possible relationship between these two factors. Injection of ethanol and N-demethyl-chlordiazepoxide (NDCDP) simultaneously (the latter being a metabolite of CDP) also elicited a more than additive depression of cerebellar c-GMP levels at 4 hr. These data suggest that NDCDP or its metabolite could be responsible for the supra-additive effect of CDP on the ethanol-induced decrease in cerebellar c-GMP levels.

Potentiation of 1-β- d-arabinofuranosylcytosine in hepatoma cells by 2′-deoxyadenosine or 2′-deoxyguanosine

A 2-hr pretreatment with 2′-deoxyadenosine (AdR), 2′-deoxyguanosine (GdR), or thymidine (TdR) synergized the growth-inhibitory effect of subsequent arabinosylcytosine (Ara-C) treatment in rat hepatoma cell lines N 1S 1 and 3924A and in the rat fibroblast line BF5. In addition, the sequence of 100 μM AdR or GdR for 2 hr followed by 15 μM Ara-C for 6 hr resulted in reduction of N 1S 1 and 3924A colony formation to 13 per cent of control values, representing synergistic killing when compared with treatment by any of these agents alone. Measurements in N 1S 1 hepatoma cells showed that AdR caused increased dATP (221 per cent) and decreased dCTP and dTTP (23 and 41 per cent respectively) in comparison with untreated controls. GdR caused increases in dGTP (351 per cent control) and dATP (191 per cent control) and decreased dCTP (36 per cent control) and dTTP (37 per cent control). Pretreatment with AdR (but not GdR) caused increased cellular Ara-CTP concentration. Pretreatment with AdR (100 μM) for 2hr increased the incorporation of [ 3H]Ara-C into DNA by 2-fold, and 2-hr pretreatment with GdR (100 μm) increased [ 3H]Ara-C incorporation into DNA 3-fold. AdR also increased incorporation of [ 3H]Ara-C into RNA, but GdR did not have this effect. In all three cell lines, AdR treatment decreased the concentrations of cellular UTP and CTP. Of the three deoxynucleosides, AdR, GdR and TdR, the most potent synergism with Ara-C was given by GdR. It was concluded that for all three nucleosides the potentiation was primarily a consequence of decreased cellular pools of dCTP.