Anxiety stress and nociceptive responses in mice.

Nociception in laboratory animals appears to be influenced by physical or emotional stressors. Nevertheless, the reported data are not univocal. Discrepancies seem to be caused by some kind of stress model and/or by the timing of stressor application. The aim of the present work is to study the influence of chronic application of a well-controlled and defined anxiety stress paradigm (rotational stress) on the behavioral formalin pain responses in mice maintained in a low-stress environment. The results indicate that emotional chronic stress increases specific pain responses in the late inflammatory phase and, correspondingly, decreases self-grooming. Locomotor activity appears influenced by pain presence only. The hormonal and neural mechanisms that could be involved in the observed nonspecific and specific nociceptive responses to stress are discussed.

Diurnal variations in tonic pain reactions in mice.

Diurnal changes in the behavioural reactions to subcutaneous formalin injection (20 microl, 1%) into the dorsum of an hindpaw were examined in female CBA/J mice aged 70-75 days, maintained in a 12/12 dark/ light cycle (light on at 07.00 h; light off at 19.00 h). Mice showed higher pain scores, as expressed by the amount of time spent licking the injected paw and by the number of flinching episodes, when tested under red light at the beginning of the dark phase (19.00-22.00: Dark group) than when tested either under white or red light at the beginning of the light phase of the diurnal cycle (7.00-10.00). The increases in pain reactions at night were found both during the first (0-10 min) and the second (11-55 min) phase of the behavioural response to formalin injection. They were not due to aspecific increases in motor behaviour, since self-grooming actually decreased in the Dark group during the second phase of the response, and the amount of locomotor activity after the injection was similar to, or lower than, that found in mice tested in the morning under white or red light, respectively. In another group of female CBA/J mice tested in the hotplate apparatus (at a temperature of 52 degrees), paw-lick latencies were significantly higher in mice tested at dark during the night, whereas jump (escape) latencies were higher in the morning. These results demonstrate different diurnal variations in the reactions to brief or prolonged noxious stimulation in mice, with greater responses to tonic pain at the onset of the dark phase.

Antimetastatic action of a new analog of dacarbazine in mice bearing Lewis lung carcinoma.

We report the selective antimetastatic properties of 3-(3,3-dimethyl-1-triazenyl)-5-methyl-4,5-dihydroisoxazole in the murine transplantable tumor model Lewis lung carcinoma. The compound verifies a previous study on the correlation of antimetastatic, antitumor and cytotoxic properties of aryl- and heteroaryltriazenes with their Electron Ionization Mass Spectrometry (EI-MS) behavior. The new analog of dacarbazine exhibits a selective antimetastatic activity accompanied by limited thymus toxicity. The mechanism of action is unclear nevertheless any antiproliferative or cytotoxic effect is excluded.

Stress and chemotherapy. Combined effects on tumor progression and immunity in animal models.

In mice bearing Lewis lung carcinoma, rotational and restraint stress specifically increases the formation of lung metastasis, and restraint stress markedly attenuates the antitumor effects of cyclophosphamide. The aim of this investigation was therefore to examine the effects of restraint stress on tumor metastasis in mice bearing MCa mammary carcinoma, and on the effectiveness of CCNU and DTIC. Restraint stress increases MCa mammary carcinoma metastasis, causes a marked reduction in cyclophosphamide activity, and a minor attenuation of the effects of CCNU and DTIC. The possible occurrence of seasonal factors, observed for the increase by rotational stress of Lewis lung carcinoma metastasis, was also determined for cyclophosphamide effectiveness. The survival time of control mice is longer in February than in June, and is not appreciably modified by rotational stress. The effects of cyclophosphamide are similar in both seasonal periods, and are similarly attenuated by rotational stress. The seasonal effects of rotational stress, and the reduction of the effects of cyclophosphamide caused by rotational stress, are accompanied by corresponding variations in the number of CD3+ and CD4+ splenic T-lymphocyte subsets and in the CD4+/CD8+ ratio, respectively. The reported effects of stress on tumor progression and on the effectiveness of cyclophosphamide thus appear to occur via modulation of immune responses of the host directed against the tumor. These data appear of interest for their experimental implications, and suggest the opportunity to consider the role that the stress during treatment may play in determining the effectiveness of clinical antitumor chemotherapy.

Melatonin administration in tumor-bearing mice (intact and pinealectomized) in relation to stress, zinc, thymulin and IL-2.

Melatonin (MEL) may counteract tumors through a direct oncostatic role. MEL is also an antistress agent with immunoenhancing properties against tumors due to a suppressive role of MEL on corticosterone release. Rotational stress (RS) (spatial disorientation) facilitates metastasis progression in mice. Also, MEL counteracts tumors because of its influence on immune responses via the metabolic zinc pool, which, is reduced in tumors and stress. Zinc is required for normal thymic endocrine activity (i.e. thymulin) and interleukin-2 (IL-2) production. Because in vivo data is still controversial, exogenous MEL treatment (22 days in drinking water) in both intact and pinealectomized (px) mice bearing Lewis lung carcinoma leads to significant decrements of metastasis volume, restoration of the negative crude zinc balance, recovery of thymulin activity and increment of IL-2 exclusively in intact and px tumor bearing mice subjected to RS. Significant inverse correlations are found in both stressed intact and px tumor bearing mice after MEL treatment between zinc and corticosterone (r = 0.78, P < 0.01; r = 0.80, P < 0.01, respectively). Positive correlations between zinc and IL-2 (r = 0.75, P < 0.01; r = 0.73, P < 0.01, respectively) or thymulin (r = 0.75, P < 0.01; r = 0.82, P < 0.01, respectively) are observed in same models of mice. These findings suggest a MEL action to decrease metastasis mediated by a possible interplay between zinc and MEL, via corticosterone, with consequent restoration of thymic efficiency and IL-2 production. MEL as an antistress agent with immunoenhancing properties in cancer deserves further consideration.nuclear factor-kb; POMC, proopiomelanocortin; Px, pinealectomized mice; RIA, radioimmunoassay; RS, rotational stress; SDI, stressed intact mice; SDPx, stressed pinealectomized mice; TNF-alpha, tumor necrosis factor-alpha; ZnFTS, active zinc-bound thymulin; ZnFTS + FTS, total thymulin.

Melatonin and oxidative damage in mice liver induced by the prooxidant antitumor drug, adriamycin.

Antioxidant properties have been attributed to melatonin; it seemed therefore worthwhile to determine its effects in relation to the prooxidant action of adriamycin, which contributes to its toxic and therapeutic effects. Melatonin effectively acts as a direct free radical scavenger in the concentration range of 20-100 microM as determined in vitro, using Fenton reaction as a source of free radicals that were determined by EPR using spin trapping method. Following the administration of a single i.v. dose of 28 mg/Kg or of 3 repeated i. p. doses of 5 mg/Kg adriamycin to CBA mice, glutathione levels in the liver cells were significantly reduced. When the treatment with adriamycin was preceded by the s.c. administration of 2 mg/Kg melatonin, the decrease in total and reduced glutathione concentrations was significantly prevented. A significant increase in lipid peroxidation was observed in liver cells after a single administration of adriamycin which was not attenuated by pretreatment with melatonin. These results indicate that further examination of the possible protective action of melatonin on the toxic effects of prooxidant antitumor drugs on normal and neoplastic tissues would be of interest also in relation to their chronotoxicological properties.

Melatonin decreases bone marrow and lymphatic toxicity of adriamycin in mice bearing TLX5 lymphoma.

When CBA male mice bearing TLX5 lymphoma were treated in the evening with a single i.v. dose of adriamycin (20-40 mg/Kg), the administration of a single pharmacological dose of melatonin (10 mg/kg s.c.) 1 hr earlier reduced the acute mortality from 10/24 to 2/24. The increase in survival time caused by adriamycin over drug untreated controls was not reduced by melatonin. The administration of melatonin alone did not cause any antitumor or evident toxic effect. Melatonin also attenuated the reduction caused by adriamycin in the number of bone marrow GM-CFU, and of CD3+, CD4+ and CD8+ splenic T-lymphocyte subsets. Reduced and total glutathione levels were decreased in the bone marrow and in the liver cells of the animals treated with adriamycin, and were significantly restored by melatonin. Moreover, lipid peroxidation by adriamycin was reduced by melatonin, as indicated by malondialdehyde measurement in the liver of the treated animals. These data indicate that the protective effects of melatonin against the host toxicity of the prooxidant antitumor drug, adriamycin, might be attributed at least partially to its antioxidant properties. These findings appear of interest in relation to the physiological rhythmic levels of endogenous melatonin and to the chronotoxicology of anthracyclines.

Seasonal effects of rotational stress on Lewis lung carcinoma metastasis and T-lymphocyte subsets in mice.

Rotational stress specifically increases the formation of spontaneous lung metastasis in mice bearing Lewis lung carcinoma, without significantly modifying the growth of primary tumor. The increase in metastasis number and volume caused by rotational stress varies in magnitude with a highly significant circannual rhythm; the acrophase approximately coincides with summer solstice. Rotational stress causes a significant reduction in the number of CD3+ and CD4+ T-lymphocyte subsets in summer, whereas in winter the number of CD3+ subset is significantly increased; the CD4+/CD8+ ratio and the number of NK 1.1 antigen positive cells are not significantly modified by rotational stress in both periods considered. The increase in metastasis formation by rotational stress thus appears to negatively correlate with the number of splenic CD3+ and CD4+ T-lymphocyte subsets. This seasonal behavior occurs in spite of the control of light cycle, temperature and humidity in the animal housing, suggesting the existence in the host of an endogenous oscillator with a circannual period. These data indicate the opportunity to consider endogenous rhythms within the host, as well as seasonal factors, in studies on stress and neuroimmunomodulation in experimental oncology.

Restraint stress reduces the antitumor efficacy of cyclophosphamide in tumor-bearing mice.

Treatment with the cytotoxic antitumor drug cyclophosphamide is highly effective in mice bearing Lewis lung carcinoma, causing the absence of macroscopically detectable tumors at necroscopy after sacrifice. When the effects of the treatment on survival are determined, a significant increase in survival time and in the proportion of long-term survivors is observed. When restraint stress is further applied, tumors develop in all of the mice treated with cyclophosphamide, and survival time and the fraction of long-term survivors are significantly reduced. Flow cytometry of splenic T-lymphocyte subsets in normal mice indicates a significant decrease in the number of CD3+, CD4+, and CD8+ subsets after treatment with cyclophosphamide and after application of restraint stress; the interaction of the two treatments is significant for CD3+ and marginally significant for CD4+ subsets. The attenuation by restraint stress which was observed for the effects of cyclophosphamide on the presence of tumors at necroscopy and for the survival of the treated mice might thus be interpreted as follows: restraint stress attenuates the immune functions of the host directed toward the weakly immunogenic tumor, an effect which, in the absence of restraint stress, interacts effectively with the cytotoxic action of cyclophosphamide toward tumor cells. The results obtained using this animal model thus indicate that experimental stress reduces the therapeutic efficacy of a cytotoxic antitumor drug; experimental and clinical implications are discussed.

Suppression of metastatic potential and up-regulation of gelatinases and uPA in LLC by protracted in vivo treatment with dacarbazine or razoxane.

Treatment of mouse Lewis lung carcinoma with razoxane or dacarbazine was protracted for 10 transplant generations. While the capacity of the treated tumors to grow locally in immuno-competent or in immuno-depressed hosts was retained and not significantly modified, the metastatic phenotype was eliminated when the treated tumor cells were transplanted into immuno-competent hosts. The reduction in metastatic potential was slightly less pronounced, in terms of both number and volume of metastases, when the treated tumor cells were transplanted into immuno-depressed hosts. These properties were retained after 3 transplant generations without treatment. Northern blotting and zymography of primary-tumor crude extracts revealed that treatment with either razoxane or dacarbazine for one generation approximately doubled the expression of MMP-2 and MMP-9, while lacking any effect on that of 1.0 and of 3.5 kb TIMP-2. When the treatment was maintained for 10 generations, the expression of MMP-2 and MMP-9 for both drugs showed up-regulation of approximately 10- and 2-fold respectively. TIMP-2 mRNA of 1.0 kb doubled its expression, while that of 3.5 kb registered just above the control. Dacarbazine doubled the expression of uPA after 10 generations, while razoxane boosted it approximately 3-fold after either 1 or 10 generations. The permanent loss of metastatic phenotype induced in Lewis lung carcinoma by dacarbazine and razoxane is thus attributable to biological mechanisms independent of down-regulation of expression and/or activation of the 2 gelatinases.

Survival time in mice bearing TLX5 lymphoma subjected to rotational stress and chemotherapy with CCNU.

The effects of a psychological stress model rotational stress were examined in mice bearing TLX5 lymphoma. The survival time of the animals was determined as a function of tumor inoculum size and treatment with the antitumor drug, CCNU. Rotational stress significantly decreased the mean survival time of mice implanted with 10 or 10(2) tumor cells, and significantly increased tumor takes in mice implanted with 10 cells. Treatment with CCNU significantly prolonged the survival time of the treated animals; the application of rotational stress significantly attenuated the increase in survival time caused by CCNU. These results indicate that in mice with a limited tumor burden, psychological stress favors the progression of TLX5 lymphoma, and reduces the effectiveness of the antitumor drug, CCNU. Moreover, the experimental model employed may provide a tool useful for investigating the mechanisms involved in the sensitivity of lymphoma to psychosocial stress.

Blockers of adrenergic neurons and receptors, tumor progression and effects of rotational stress in mice.

The aim of this work was to determine the possible participation of adrenergic responses to the increase in tumor dissemination induced by rotational stress using drugs affecting monoaminergic function. The growth of the primary tumor and the formation of lung metastasis were determined in mice implanted with Lewis lung carcinoma, subjected to rotational stress and treated with the adrenergic neuron blocker reserpine, the alpha-receptor blocker phenoxybenzamine, and the beta-blocker propranolol. Treatment with reserpine markedly reduced the formation of spontaneous lung metastasis and completely abolished the increase in metastases caused by rotational stress without direct effect on tumor cells or blood vessels. Phenoxybenzamine and propranolol caused opposite effects on tumor progression. Previous immunosuppression by cyclophosphamide in mice with tumors reduced the antimetastatic effects of reserpine. These results suggesting the importance of the adrenergic modulation of immune resistance factors in controlling metastasis development and indicate a possible role in monitoring the use and effects of monoaminergic drugs in cancer patients.

Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins.

Micrococcal nuclease digestion of nuclei from mouse Lewis lung carcinoma cells releases a protein mixture into the supernatant that lacks histone H1 and contains a full complement of high-mobility-group I (HMGI) proteins (i.e. I, Y and I-C). This implies that all three HMGI proteins are localized at the nuclease-sensitive regions of active chromatin. It is also shown that if Ca2+ ions are present in the nuclear incubation buffer (with or without exogenous nuclease), all three HMGI proteins become ADP-ribosylated. We propose that this modification of HMGI family proteins is part of the general poly(ADP-ribosyl)ation that accompanies DNA damage in apoptosis and other processes.

Effects of prolonged treatment with decarbazine on tumor metastatic potential in mice bearing Lewis lung carcinoma.

The effects of decarbazine on tumour growth and metastatic dissemination upon treatment protracted for 10 tumour transplant generations were examined in mice bearing Lewis lung carcinoma. Primary tumour growth is unaffected by the drug, independently from the duration of the treatment. In contrast, dacarbazine significantly inhibits the formation of lung metastasis. The proportion of mice with metastasis decreases for an increasing number of transplant generations of treatment, and after 10 transplant generations of treatment metastatic capacity is completely lost in immunocompetent mice. The reduction in metastatic potential is relatively stable, being retained for three successive transplant generations without treatment. The metastatic potential of treated tumours in immunosuppressed mice is substantially similar to that in immunocompetent hosts, indicating that chemical xenogenization of tumour cells does not occur as reported for transplantable mouse leukaemias. The results obtained using clonally selected tumour lines with different metastatic potential rule out the selection by dacarbazine of tumour cell sublines with reduced metastatic potential as the mechanism of the drug's action. Upon prolonged treatment, dacarbazine appears to cause a rather stable and dramatic loss in metastatic potential, not accompanied by resistance, which might be attributed to genotypic alteration(s) of tumour cells, and which might participate into the clinical effects of the drug.

Metastasis and neuroendocrine system in stressed mice.

The effects of experimental stressors have been examined for their differential effects on primary tumor growth and spontaneous lung metastasis in syngeneic mice bearing the weakly immunogenic tumor Lewis lung carcinoma. The effects caused by the early weaning, physical restraint and foot-shock are in general small, and affected by a high variability. On the contrary, spatial disorientation reproducibly causes a specific increase in tumor metastases. The effects of spatial disorientation are sensitive to the inhibition by treatment with bromocryptine and guanethidine, and particularly by a central and peripheral adrenergic neuron blocking agent reserpine. The use of different lighting conditions and assay of urinary melatonin excretion reveal an association between pineal gland function and effects of spatial disorientation on metastasis.

Seasonal dependency of the effects of experimental stressors on tumor metastasis in mice bearing Lewis lung carcinoma.

Increasing evidence indicates that the application of stressor paradigms in experimental animals affects tumor incidence and progression. However, a high heterogeneity appears both for the animal-tumor system used and for the characteristics of the stressor employed. A high variability was observed also with the application of rotational stress, a carefully and widely characterized mild psychological stressor, to mice bearing Lewis lung carcinoma. The aim of this work has been therefore to examine the possible seasonal dependency of the effects of experimental stressors (rotational stress, forced immobilization and electric foot shock) on spontaneous lung metastasis formation in mice bearing Lewis lung carcinoma. The possible participation of pineal gland and of melatonin have also been examined including in the experimental protocol the measurement of melatonin urinary excretion. The stressor paradigms used significantly increased metastasis weight in spring, in comparison with non-stressed animals. When examined in winter, rotational stress and foot shock significantly decreased metastasis formation, in comparison with non-stressed mice. The effects of forced immobilization were not season-dependent. The melatonin urinary excretion has been measured in relation to the seasonal effects of rotational stress. Nocturnal melatonin excretion is markedly increased by rotational stress in spring and is remarkably decreased in winter. These variations in endogenous melatonin levels caused by rotational stress appear to directly correlate with the effects of the stressor or metastasis. These results lend support to the view that the mechanisms underlying the tumor enhancing action of stressors involve the psychoneuroendocrine network, and indicate the relevance of chronobiology in experimental cancer research and neuro-immuno-modulation.

A non-invasive simple method for measurement of urinary excretion of melatonin in undisturbed mice.

Melatonin is a hormone involved in neuroendocrine responses; its plasma concentrations display a circadian pattern which is modified by stress. Studies for determining the effects of stressors on melatonin levels in laboratory animals present the difficulty that the procedures for blood sampling are by themselves potential stressors capable of influencing the levels of the hormone measured. A simple non-stressful method for measuring urinary excretion of melatonin has been consequently developed. The method is applicable to single undisturbed mice kept in conventional cages, and consists of urine collection on chromatographic paper followed by extraction and melatonin assay by radioimmunoassay. The use of this method with BD2F1 mice indicates nocturnal excretion of melatonin significantly higher than during the day; nighttime melatonin levels were shown to be suppressed by constant illumination. A significant increase in nocturnal melatonin excretion was caused by the application of rotational stress applied as a mild experimental stressor.