Tricyclic antidepressants and the incidence of certain cancers: a study using the GPRD.

BACKGROUND: Several studies suggest links between cancer and tricyclic antidepressant use. METHODS: A case-control study using the General Practice Research Database examined whether previous tricyclic usage was associated with reduced incidence of brain (with glioma as a sub-category), breast, colorectal, lung and prostate cancers. Conditional logistic regression adjusted for age, gender, general practice, depression, smoking, body mass index, alcohol use and non-steroidal anti-inflammatory drug use. RESULTS: A total of 31 953 cancers were identified, each matched with up to two controls. We found a statistically significant reduction in tricyclic prescriptions compared with controls in glioma (odds ratio (OR) =0.59, 95% confidence interval (CI)=0.42-0.81) and colorectal cancer patients (OR=0.84, CI=0.75-0.94). These effects were dose-dependent (P-values for trend, glioma=0.0005, colorectal=0.001) and time-dependant (P-values for trend glioma=0.0005, colorectal=0.0086). The effects were cancer-type specific, with lung, breast and prostate cancers largely unaffected by antidepressant use. CONCLUSION: The biologically plausible, specific and dose- and time-dependant inverse association that we have found suggests that tricyclics may have potential for prevention of both colorectal cancer and glioma.

The effects of antidepressants on mitochondrial function in a model cell system and isolated mitochondria.

The in vitro effects of antidepressant drugs on mitochondrial function were investigated in a CHOß(2)SPAP cell line used previously to determine the effects of antidepressants on gene transcription (Abdel-Razaq et al., Biochem Pharmacol 73:1995-2003, 2007) and in rat heart isolated mitochondria. Apoptotic effects of clomipramine (CLOM), desipramine (DMI) and of norfluoxetine (NORF, the active metabolite of fluoxetine), on cellular viability were indicated by morphological changes and concentration-dependent increases in caspase-3 activity in CHO cells after 18 h exposure to CLOM, DMI and NORF. However, tianeptine (TIAN) was without effect. CLOM and NORF both reduced integrated mitochondrial function as shown by marked reductions in membrane potential (MMP) in mitochondria isolated from rat hearts. DMI also showed a similar but smaller effect, whereas, TIAN did not elicit any significant change in MMP. Moreover, micromolar concentrations of CLOM, DMI and NORF caused significant inhibitions of the activities of mitochondrial complexes (I, II/III and IV). The inhibitory effects on complex IV activity were most marked. TIAN inhibited only complex I activity at concentrations in excess of 20 µM. The observed inhibitory effects of antidepressants on the mitochondrial complexes were accompanied by a significant decrease in the mitochondrial state-3 respiration at concentrations above 10 µM. The results demonstrate that the apoptotic cell death observed in antidepressant-treated cells could be due to disruption of mitochondrial function resulting from multiple inhibition of mitochondrial enzyme complexes. The possibility that antimitochondrial actions of antidepressants could provide a potentially protective pre-conditioning effect is discussed.

The effects of antidepressants on cyclic AMP-response element-driven gene transcription in a model cell system.

The effects of the antidepressant drugs clomipramine (CLOM), desipramine (DMI), tianeptine (TIAN) and of norfluoxetine (NORF, the active metabolite of fluoxetine), were investigated in CHO cells expressing human beta2 adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter gene to determine their actions on cyclic AMP-driven gene transcription. After 18 h of exposure, CLOM, DMI and NORF, but not TIAN, had biphasic effects on 1 microM isoprenaline-stimulated SPAP fsproduction with concentrations between 10 nM and 1 microM enhancing the maximal (E(max)) SPAP response, without changing EC50 values, but higher concentrations produced marked inhibitory effects. At nanomolar concentrations, CLOM and DMI increased expression of phospho-CREB (cyclic AMP response element binding protein). NORF was less effective but did significantly increase phospho-CREB at a concentration of 200 nM. TIAN had no effect. None of the antidepressants had any effect on CREB expression, nor on the accumulation of cyclic AMP. After prolonged exposure (7-21 days) to a low concentration (200 nM) of the antidepressants, the enhanced E(max) values for SPAP production evident after 18 h were not maintained but CLOM and DMI induced a significant leftward shift in the isoprenaline EC50 after a 7-day period of treatment and this was sustained at the 21 day time point. TIAN did not produce any significant changes. The results demonstrate that, in vitro, some but not all antidepressants can modify gene transcription via monoamine and cyclic AMP-independent mechanisms. The in vivo adaptive responses to TIAN probably involve alterations in different gene sets to those affected by other antidepressants.

Metabolites in the developing rat liver--a proton nuclear magnetic resonance spectroscopic study.

We have used 1H-NMR spectroscopy in vitro to investigate metabolite changes in the rat liver in the first 21 days of life. The principle findings are firstly that betaine, a metabolite of choline, was relatively low (1-2 mumol/g) on days 1-7, then rose sharply to 5-6 mumol/g by day 19, whereas approximately reciprocal changes occurred in taurine levels. Secondly the lactate levels were remarkably low (0.1-0.8 mumol/g) on days 1-7. Changes in two other choline derivatives, phosphocholine (PC) and glycerophosphorylcholine (GPC) are also reported. The results are discussed in the context of the origin of these metabolites in the neonatal period, their levels in the adult (180 day-old) rat and the significance of the measured changes in metabolite levels during liver development.

Endoplasmic reticulum: the major contributor to the PDE peak in hepatic 31P-NMR spectra at low magnetic field strengths.

31P-NMR spectra of liver in vivo, subcellular fractions and model systems were acquired in order to characterise further the hepatic phosphodiester peak seen at low magnetic field strengths previously shown to be predominantly due to phospholipid bilayers. The data obtained in this study in vitro suggested that the phospholipid membranes of the endoplasmic reticulum provide the dominant contribution to this phosphodiester peak. Support for this hypothesis was provided by experiments on rats. Phenobarbitone, which is known to induce proliferation of the endoplasmic reticulum produced a considerable increase in intensity of the phosphodiester peak in liver spectra in vivo.

The effects of desmethylimipramine on cyclic AMP-stimulated gene transcription in a model cell system.

The present study utilised an in vitro cell model of the cAMP signalling pathway to investigate the actions of desipramine (DMI) and other psychoactive agents on cAMP-driven gene transcription. The model comprised CHObeta2 SPAP cells; Chinese hamster ovary cells expressing human beta2 adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter gene with multiple cAMP response elements (CREs) in its promoter region. SPAP assays showed DMI to inhibit isoprenaline or forskolin-enhanced gene transcription in a time and concentration-dependent manner (IC50=16.6+/-2.0 microM after 18 h). This effect of DMI was not dependent upon activity at the levels of the beta2 receptor, cAMP accumulation or phosphorylation of the transcription factor, cAMP response element binding protein (CREB). The inhibitory effects were maintained in the presence of DMI for at least 3 weeks and were mimicked by exposure to norfluoxetine (the major metabolite of fluoxetine; IC50=7.2+/-1.8 microM) and the neuroleptics, chlorpromazine and clozapine, all at a concentration of 10 microM. Amphetamine (10 microM, 18 h) enhanced SPAP gene transcription. Ca2+ imaging experiments ruled out an inhibitory effect of DMI on Ca2+ influx as concluded by previous studies. The results suggest a molecular target for DMI that lies downstream of CREB phosphorylation. Whether the inhibitory action of DMI is common to naturally expressed CRE-driven genes involved in adaptive responses to antidepressants in vivo remains to be determined.

Activity of mitochondrial respiratory chain enzymes after transient focal ischemia in the rat.

Previous results demonstrated that after 2-hour middle cerebral artery occlusion (MCAO) in the rat, 1- to 2-hour recirculation temporarily restored the bioenergetic state and mitochondrial function, but secondary deterioration took place after 4 hours. The authors measured the activity of mitochondrial respiratory chain complexes, citrate synthase, and glutamate dehydrogenase as possible targets of secondary damage. Focal and penumbral tissues were sampled in the control condition, after 2 hours of MCAO, and after 1, 2, or 4 hours of postischemic recirculation; two groups were treated with alpha-phenyl-N-tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.

beta-Amyloid fragment 25-35 selectively decreases complex IV activity in isolated mitochondria.

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been demonstrated in Alzheimer's disease, and after the expression of the amyloid precursor protein (APP) in cultured cells, suggesting that mitochondria might be involved in beta-amyloid toxicity. Recent evidence suggests that the proteolysis of APP to generate beta-amyloid is at least in part intracellular, preceding the deposition of extracellular fibrils. We have therefore investigated the effect of incubation of isolated rat brain mitochondria with the beta-amyloid fragment 25-35 (100 microM) on the activities of the mitochondrial respiratory chain complexes I, II-III, IV (cytochrome c oxidase) and citrate synthase. The peptide caused a rapid, dose-dependent decrease in the activity of complex IV, white it had no effect on the activities on any of the other enzymes tested. The reverse sequence peptide (35-25) had no effect on any of the activities measured. We conclude that inhibition of mitochondrial complex IV might be a contributing factor to the pathogenesis of Alzheimer's disease.

Time-dependent impairment of mitochondrial function after storage and transplantation of rabbit kidneys.

BACKGROUND: The mitochondrial respiratory chain is implicated as a major target of kidney damage after ischemia-reperfusion. This study measures changes in integrated mitochondrial function and in the activity of enzymes of the respiratory chain after cold storage and transplantation-reperfusion in vivo. METHODS: Mitochondrial oxygen consumption and activities of respiratory chain enzymes and citrate synthase were measured in cortical mitochondria isolated from rabbit kidneys after 1-48 hr of cold ischemia with or without transplantation-reperfusion. RESULTS: State 4 mitochondrial oxygen consumption was significantly increased after 48 hr of ischemia or 24-48 hr of ischemia with transplantation. Prolonged (24 or 48 hr) ischemic storage with and without transplantation caused a significant decrease in state 3 oxygen consumption, as did transplantation after 1, 24, and 48 hr of cold storage. Complex I and complex II-III activity decreased after 24 or 48 hr of ischemia, with transplantation having little additional effect. Complex IV activity was significantly decreased after 48 hr of ischemia, this decrease being exacerbated by transplantation-reperfusion. Complex V activity decreased significantly after 1 hr of ischemia and continued to decrease after 24-48 hr of ischemia. Transplantation after 1-24 hr (but not 48 hr) of ischemia resulted in partial recovery of complex V activity. Citrate synthase activity was decreased significantly only after 48 hr of ischemia and reperfusion, consistent with the loss of mitochondrial membrane integrity seen in electron micrographs of the transplanted 48-hr group. CONCLUSIONS: These data suggest that individual rabbit kidney mitochondrial complexes have different susceptibilities to cold ischemic and reperfusion damage.

Ammonia causes a drop in intracellular pH in metabolizing cortical brain slices. A [31P]- and [1H]nuclear magnetic resonance study.

[31P]- and [1H]Nuclear magnetic resonance spectroscopy were used to study metabolism in cortical brain slices in the guinea-pig during acute exposure to pathophysiological concentrations of ammonia. Intracellular acidification, measured from the chemical shift of endogenous inorganic phosphate, was observed without any change in cellular energy status or concentrations of lactate, glutamate and glutamine. The initial acidification, which developed over a period of 9 min appeared to be heterogeneous, on the basis of a splitting of the inorganic phosphate resonance in a number of experiments, corresponding to pH changes of 0.07 and 0.27 pH units. Subsequently a homogeneous acidification, of 0.15 pH units, developed by 23 min following exposure to ammonia. Intracellular pH recovered within 6 min after discontinuation of the ammonia load. In the absence of external bicarbonate, intracellular pH was 0.12 units more acidic than in the bicarbonate buffer and ammonia caused a further acidification by 0.16 units. When glutamine synthase inhibitor, methionine sulphoximine, was added, there was a slow fall in intracellular pH. Under these conditions, subsequent addition of ammonia failed to cause acidification directly. Thus acute elevation of ammonia does not lead to a change in cerebral high-energy phosphate or lactate metabolism, but may be associated with a fall in cortical intracellular pH.

Effect of hydrazine upon vitamin B12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes.

The effect of the industrial chemical, hydrazine (4-12 mM), on methionine synthase (EC 2.1.1.13) activity and levels of the sulphur amino acids homocysteine, cysteine, and taurine as well as GSH were investigated in vitro in isolated rat hepatocyte suspensions and monolayers in order to explain some of the adverse in vivo effects of hydrazine. None of the concentrations of hydrazine were overtly cytotoxic in hepatocyte suspensions (measured as lactate dehydrogenase [LDH] leakage) after 3 hr. However, after 24 hr in culture cells treated with 12 mM, hydrazine showed a significant increase in LDH leakage. Methionine synthase activity was reduced by hydrazine (8 and 12 mM) in suspensions (by 45 and 55%, after 3 hr) and monolayers (12 mM; 65-80% after 24 hr). This was not due to nitric oxide production and the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine, failed to protect against the hydrazine-induced loss of ATP and GSH and the reduction in urea synthesis at 24 hr. Homocysteine export was increased by 6 mM hydrazine, and total taurine content of treated cells was increased by 12 mM hydrazine. Thus, hydrazine was found to have several important and possibly deleterious effects on some parts of the sulphur amino acid pathway.

[U-13C]glutamate metabolism in rat brain mitochondria reveals malic enzyme activity.

13C nuclear magnetic resonance spectroscopy was used to study the activity of malic enzyme in isolated brain mitochondria from rat in the presence of unlabelled malate and [U-13C]glutamate. ADP, inorganic phosphate, malate and [U-13C]glutamate were added to a suspension of oxygenated mitochondria. Typical tricarboxylic acid (TCA) cycle constituents (malate, 2-oxoglutarate and succinate) were labelled from [U-13C]glutamate and detected in the superfusion medium. The labelling patterns in the different atom positions of glutamate revealed entry of both unlabelled and labelled acetyl-CoA into the TCA cycle. Unlabelled acetyl-CoA was derived via pyruvate from exogenously applied malate by the action of mitochondrial malic enzyme, while labelled acetyl-CoA was derived from TCA cycle intermediates, most likely by the action of mitochondrial malic enzyme on malate produced from [U-13C]glutamate. The results demonstrate malic enzyme activity and pyruvate recycling in isolated rat brain mitochondria.

Inhibition of N-acetylaspartate production: implications for 1H MRS studies in vivo.

The effect of specific irreversible inhibitors of complexes I, III, IV and V of the mitochondrial respiratory chain, (rotenone, myxothiazol, cyanide and oligomycin, respectively) on mitochondrial N-acetylaspartate production, and its relationship to oxidative phosphorylation (ATP production and oxygen consumption) were investigated in isolated rat brain mitochondria. Mitochondrial N-acetylaspartate production, ATP production and oxygen consumption were all significantly decreased in the presence of each of the inhibitors used compared with control incubations, and correlated positively with each other. It is postulated that decreased N-acetylaspartate levels seen in disease states by 1H NMR spectroscopy in vivo may reflect primarily an impaired mitochondrial energy production rather than neuronal cell loss.

Heightened resistance of the neonatal brain to ischemia-reperfusion involves a lack of mitochondrial damage in the nerve terminal.

Mitochondria are known targets of ischemia-reperfusion injury in adult brain. Although neonates are more resistant to ischemic episodes, the mechanisms accounting for this are not yet fully understood. The aim of this study therefore was to determine whether a difference in post-ischemic mitochondrial function may play a role in the heightened recovery of the neonatal brain following ischemia-reperfusion. We have therefore compared the effects of an in vitro model of ischemia on the enzymes of the mitochondrial respiratory chain in isolated nerve terminals (synaptosomes) from neonatal and adult rats. Ischemia caused a significant, reversible decrease in mitochondrial Complex I activity in both adult and neonatal preparations. In neonatal preparations alone, ischemia also led to a significant decrease in mitochondrial Complexes II-III activity. Following 30 min of reperfusion mitochondrial Complexes II-III and IV exhibited decreased activity in synaptosomes from adult, but not neonatal rats. These data suggest a difference in the susceptibility of adult as compared to neonatal nerve terminal mitochondria to ischemia-reperfusion. These data show for the first time that nerve terminal mitochondria from immature animals remain undamaged following a period of ischemia and reperfusion, in contrast to nerve terminal mitochondria from the adult brain. This adds to the growing body of evidence that mitochondrial function plays a key role in neuronal death following cerebral ischemia reperfusion.

Effect of postischaemic hypothermia on the mitochondrial damage induced by ischaemia and reperfusion in the gerbil.

In order to test the effect of hypothermia on mitochondrial function damage following cerebral ischaemia/reperfusion, Mongolian gerbils were submitted to 30 min bilateral carotid occlusion and 2 h of reperfusion at 37 degreesC or 30 degreesC. After normothermic (37 degreesC) ischaemia/reperfusion, significant decreases in mitochondrial state 3 (+ADP) oxygen consumption (-42.2%), complex II-III activity in synaptosomes (-31.7%) and complex IV were measured, in both free mitochondria and synaptosomes (-30.3% and -27. 8% respectively). However, following hypothermic (30 degreesC) reperfusion, both respiration rates and all enzyme activities remained at levels not significantly different from those in the sham operated controls.

Effect of fish oil on cancer cachexia and host liver metabolism in rats with prostate tumors.

The aim of this study was to investigate whether tumor-induced cachexia and aberrations in host liver metabolism, induced by the MAT-LyLu variant of the Dunning prostate tumor, could be prevented by omega 3 fatty acids from fish oil. On day 0, adult Copenhagen-Fisher rats fed normal chow ad libitum were inoculated with 10(6) MAT-LyLu cells (n = 14) or saline (n = 9). On day 7, when tumors were palpable, four tumor-bearing (TB) and four nontumor-bearing (NTB) rats were put on isocaloric diets with 50% of total energy as fish oil. The introduction of fish oil-enriched diets caused a reduction in energy intake to less than half of the energy intake by animals fed normal diets during days 7-14 (difference by dietary group: NTB, P < 0.001; TB, P < 0.001). During days 14-21, energy intake in fish oil-fed animals returned to approximately 75% of energy intake by animals fed normal diets (difference by dietary group: NTB, P < 0.003; TB, P = 0.001). Carcass weight of animals on day 21, when the study was terminated, was significantly related to initial weight (P = 0.05) and mean food intake during the study (P = 0.01). When data were adjusted for these variables using analysis of covariance, with NTB animals on normal diets being the reference group, significant loss of carcass weight was observed in TB animals on normal diets only (mean +/- SEM 58 +/- 10 g loss, P < 0.001), but not in TB animals on fish oil diets (8 +/- 18 g loss, P = 0.67).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of landfill leachate irrigation on red maple (Acer rubrum L.) and sugar maple (Acer saccharum Marsh.) seedling growth and on foliar nutrient concentrations.

Greenhouse experiments were conducted to investigate the nature and severity of stresses imposed on northern hardwood tree species (red maple (Acer rubrum L.) and sugar maple (Acer saccharum Marsh.)) by the application of municipal landfill leachate. Red maple seedlings received applications of untreated and pretreated (lime, activated carbon) leachate, to both leaves and soil, at irrigation rates consistent with evapotranspirational demands. Plant height measurements indicated no significant growth effects arising from leachate application over a 7-week period. Stem diameter, however, was positively affected by applications of both untreated and lime-treated leachate diluted to 75% with deionized water. Iron foliar concentrations were significantly higher in seedlings irrigated with untreated leachate applied to leaves and soil, but not in seedlings where leachate was applied to soil only. Nitrogen foliar concentrations were substantially higher in seedlings receiving undiluted and untreated leachate applied to the soil only. The Cu concentration of the red maple foliage decreased appreciably in plants receiving moderate applications of leachate. Foliar Ca concentrations decreased notably in seedlings irrigated with untreated leachate applied to the soil and with diluted, carbon-treated leachate. The Cu concentration of the red maple foliage decreased appreciably in plants receiving applications of undiluted and 50% water-diluted lime-treated leachate while Mn levels were consistently high across all treatments. Leachate application did not cause any discernable changes in foliar concentrations of P, K, Mg, B or Zn. In an ancillary experiment, sugar maple seedlings were subjected to saturation/ drainage treatment cycles with undiluted and untreated leachate. Severe visible symptoms of vegetative stress were apparent within 24 h and 100% seedling mortality occurred after five such waterlogging cycles. Fe assimilation was apparent in both leachate treatments relative to the 24 h water treatment. Despite the short-term nature of the experiments, the results indicate how quickly forest vegetation may respond to altered chemical environments. This underscores the need for correct installation and control of leachate irrigation systems.

Regulation of heat shock protein synthesis in human skin fibroblasts in response to oxidative stress: role of vitamin E.

Skin plays an important role in protection against oxidative stressors such as ultraviolet radiation, ozone and chemicals. Chronic sun exposure causes degenerative changes in the skin that are recognized as photoaging. Oxidative stress has been shown to alter the expression of mammalian antioxidant enzymes as well as to enhance numerous transcription factors, including nuclear factor kappaB, stress-activated protein kinase and heat shock factor This latter is the transcription factor for the synthesis of heat shock proteins, which have been known to protect against a wide variety of toxic conditions, including extreme temperatures, oxidative stress and cytotoxic drugs. In this study we investigated the role of oxidative stress in the induction of heat shock protein (HSP) 70 in human skin fibroblasts and the effect of vitamin E. We found that significant HSP70 induction occurred after exposure to HOOH and that this was associated with a significant perturbation in protein and nonprotein sulfhydryl groups, and with a significant increase in protein carbonyl levels. Treatment with vitamin E conferred significant protection against stress-induced modifications of cellular sulfhydryl and carbonyl content, while maintaining functional levels of cytoprotective HSP70. Our results point to the possible involvement of redox mechanisms in the heat shock signal transduction pathway, which may play an important regulatory role in the genetic mechanisms of tolerance to oxidative stress. Exogenous antioxidant supplementation with vitamin E could have cosmetic benefits and may be an efficient tool to mitigate the consequences of free radical-induced skin damage.

Induction of heat shock protein synthesis in human skin fibroblasts in response to oxidative stress: regulation by a natural antioxidant from rosemary extract.

Reactive oxygen species have been implicated in the pathogenesis of the severe connective tissue damage present in several photodermatologic disorders, including drug-induced phototoxicity, porphyrias and photoaging. Oxidative stress has been shown to alter the expression of mammalian antioxidant enzymes and to enhance numerous transcription factors, including nuclear factor-kappa B, stress-activated protein kinase and heat shock factor. The latter represents the transcription factor for the synthesis of cytoprotective proteins called heat shock proteins. In this study, we investigated the role of oxidative stress and sulfdryl (SH) groups in the induction of HSP70 in human skin fibroblasts and the effect of antioxidants. We found that significant HSP70 induction occurred after exposure to HOOH and this was associated with marked perturbation in protein and nonprotein SH groups and with a considerable increase in protein carbonyl levels. Treatment with a natural antioxidant from rosemary extract provided notable protection against stress-induced modifications of cellular SH and carbonyl content, maintaining functional levels of cytoprotective heat shock protein 70. Our results point to the possible involvement of redox mechanisms in the heat shock signal transduction pathway, which may play an important regulatory role in the genetic mechanisms of tolerance to oxidative stress. Exogenous supplementation of an antioxidant hydrophilic extract from rosemary could have cosmetic benefits and may represent an efficient tool to minimize free radical-induced skin damage.

Chlorimipramine: a novel anticancer agent with a mitochondrial target.

Mitochondria have been suggested to be a potential intracellular target for cancer chemotherapy. In this report, we demonstrate the ability of the tricyclic antidepressant chlorimipramine to kill human glioma cells in vitro by a molecular mechanism resulting in an increase in caspase 3 activity following inhibition of glioma oxygen consumption. Studies with isolated rat mitochondria showed that chlorimipramine specifically inhibited mitochondrial complex III activity, which causes decreased mitochondrial membrane potential as well as mitochondrial swelling and vacuolation. The use of chlorimipramine in human as an effective, non-toxic cancer therapeutic having a strong selectivity between cancer cells and normal cells on the basis of their mitochondrial function is discussed.