Serotonergic modulation of pain and analgesic responses: a study in rats with constitutionally altered serotonin transporters.

BACKGROUND: A role of the serotonin (5HT) transporter, a key regulator of serotonergic transmission, in the physiology, pharmacology and genetics of pain responses has been proposed recently. The present study aimed to explore the impact of constitutive differences in the activity of the serotonin transporter, and 5HT homeostasis in general, on the modulation on pain sensitivity and analgesic responses to drugs that utilize 5HT mechanisms. METHODS: A novel genetic animal model, Wistar-Zagreb 5HT rats, obtained by selective breeding of animals for extreme activity of the platelet serotonin transporter was used. As a consequence of breeding, two sublines of this model, termed high-5HT and low-5HT, differ in both central and peripheral serotonin homeostasis. Thermal pain sensitivity of 5HT sublines was assessed at baseline and following administration of analgesic drugs, as determined by paw withdrawal latency to radiant heat stimulation. RESULTS: Animals from 5HT sublines show differences in both basal pain sensitivity and analgesic responses. Rats with the low-5HT phenotype displayed decreased baseline paw withdrawal latencies (hyperalgesia) in comparison to their high-5HT counterpart (25%; p < 0.001). They also showed better analgesic response to acute and prolonged treatment with tramadol (p = 0.027) and clomipramine (p = 0.019), respectively, whereas administration of fluvoxamine did not produce an analgesic effect in either 5HT subline. CONCLUSIONS: These findings support the idea that functionality of the serotonin transporter is one of the physiological/genetic determinants of individual differences in pain responses and modulation. They also validate Wistar-Zagreb 5HT rats, with constitutionally up-regulated/down-regulated serotonin transporter, as a potential new genetic model for studying serotonergic modulation of pain responses.

Lipid peroxidation, detoxification capacity, and genome damage in mice after transplacental exposure to pharmaceutical drugs.

Data on genome damage, lipid peroxidation, and levels of glutathione peroxidase (GPX) in newborns after transplacental exposure to xenobiotics are rare and insufficient for risk assessment. The aim of the current study was to analyze, in an animal model, transplacental genotoxicity, lipid peroxidation, and detoxification disturbances caused by the following drugs commonly prescribed to pregnant women: paracetamol, fluconazole, 5-nitrofurantoin, and sodium valproate. Genome damage in dams and their newborn pups transplacentally exposed to these drugs was investigated using the in vivo micronucleus (MN) assay. The drugs were administered to dams intraperitoneally in three consecutive daily doses between days 12 and 14 of pregnancy. The results were correlated, with detoxification capacity of the newborn pups measured by the levels of GPX in blood and lipid peroxidation in liver measured by malondialdehyde (HPLC-MDA) levels. Sodium valproate and 5-nitrofurantoin significantly increased MN frequency in pregnant dams. A significant increase in the MN frequency of newborn pups was detected for all drugs tested. This paper also provides reference levels of MDA in newborn pups, according to which all drugs tested significantly lowered MDA levels of newborn pups, while blood GPX activity dropped significantly only after exposure to paracetamol. The GPX reduction reflected systemic oxidative stress, which is known to occur with paracetamol treatment. The reduction of MDA in the liver is suggested to be an unspecific metabolic reaction to the drugs that express cytotoxic, in particular hepatotoxic, effects associated with oxidative stress and lipid peroxidation.

Lipid peroxidation, detoxification capacity, and genome damage in mice after transplacental exposure to pharmaceutical drugs

Data on genome damage, lipid peroxidation, and levels of glutathione peroxidase (GPX) in newborns after transplacental exposure to xenobiotics are rare and insufficient for risk assessment. The aim of the current study was to analyze, in an animal model, transplacental genotoxicity, lipid peroxidation, and detoxification disturbances caused by the following drugs commonly prescribed to pregnant women: paracetamol, fluconazole, 5-nitrofurantoin, and sodium valproate. Genome damage in dams and their newborn pups transplacentally exposed to these drugs was investigated using the in vivo micronucleus (MN) assay. The drugs were administered to dams intraperitoneally in three consecutive daily doses between days 12 and 14 of pregnancy. The results were correlated, with detoxification capacity of the newborn pups measured by the levels of GPX in blood and lipid peroxidation in liver measured by malondialdehyde (HPLC-MDA) levels. Sodium valproate and 5-nitrofurantoin significantly increased MN frequency in pregnant dams. A significant increase in the MN frequency of newborn pups was detected for all drugs tested. This paper also provides reference levels of MDA in newborn pups, according to which all drugs tested significantly lowered MDA levels of newborn pups, while blood GPX activity dropped significantly only after exposure to paracetamol. The GPX reduction reflected systemic oxidative stress, which is known to occur with paracetamol treatment. The reduction of MDA in the liver is suggested to be an unspecific metabolic reaction to the drugs that express cytotoxic, in particular hepatotoxic, effects associated with oxidative stress and lipid peroxidation.

Animal model for age- and sex-related genotoxicity of diethylstilbestrol

Environmental xenoestrogens pose a significant health risk for all living organisms. There is growing evidence concerning the different susceptibility to xenoestrogens of developing and adult organisms, but little is known about their genotoxicity in pre-pubertal mammals. In the present study, we developed an animal model to test the sex- and age-specific genotoxicity of the synthetic estrogen diethylstilbestrol (DES) on the reticulocytes of 3-week-old pre-pubertal and 12-week-old adult BALB/CJ mice using the in vivo micronucleus (MN) assay. DES was administered intraperitoneally at doses of 0.05, 0.5, and 5 µg/kg for 3 days and animals were sampled 48, 72 and 96 h, and 2 weeks after exposure. Five animals were analyzed for each dose, sex, and age group. After the DES dose of 0.05 µg/kg, pre-pubertal mice showed a significant increase in MN frequency (P < 0.001), while adults continued to show reference values (5.3 vs 1.0 MN/1000 reticulocytes). At doses of 0.5 and 5 µg/kg, MN frequency significantly increased in both age groups. In pre-pubertal male animals, MN frequency remained above reference values for 2 weeks after exposure. Our animal model for pre-pubertal genotoxicity assessment using the in vivo MN assay proved to be sensitive enough to distinguish age and sex differences in genome damage caused by DES. This synthetic estrogen was found to be more genotoxic in pre-pubertal mice, males in particular. Our results are relevant for future investigations and the preparation of legislation for drugs and environmentally emitted agents, which should incorporate specific age and gender susceptibility.

Animal model for age- and sex-related genotoxicity of diethylstilbestrol.

Environmental xenoestrogens pose a significant health risk for all living organisms. There is growing evidence concerning the different susceptibility to xenoestrogens of developing and adult organisms, but little is known about their genotoxicity in pre-pubertal mammals. In the present study, we developed an animal model to test the sex- and age-specific genotoxicity of the synthetic estrogen diethylstilbestrol (DES) on the reticulocytes of 3-week-old pre-pubertal and 12-week-old adult BALB/CJ mice using the in vivo micronucleus (MN) assay. DES was administered intraperitoneally at doses of 0.05, 0.5, and 5 microg/kg for 3 days and animals were sampled 48, 72 and 96 h, and 2 weeks after exposure. Five animals were analyzed for each dose, sex, and age group. After the DES dose of 0.05 microg/kg, pre-pubertal mice showed a significant increase in MN frequency (P < 0.001), while adults continued to show reference values (5.3 vs 1.0 MN/1000 reticulocytes). At doses of 0.5 and 5 microg/kg, MN frequency significantly increased in both age groups. In pre-pubertal male animals, MN frequency remained above reference values for 2 weeks after exposure. Our animal model for pre-pubertal genotoxicity assessment using the in vivo MN assay proved to be sensitive enough to distinguish age and sex differences in genome damage caused by DES. This synthetic estrogen was found to be more genotoxic in pre-pubertal mice, males in particular. Our results are relevant for future investigations and the preparation of legislation for drugs and environmentally emitted agents, which should incorporate specific age and gender susceptibility.

The antitumor activity of thymoquinone and thymohydroquinone in vitro and in vivo.

AIM: The aim of the study was to investigate antitumor activity of thymoquinone (TQ) and thymohydroquinone (THQ) in vitro and in vivo. MATERIALS AND METHODS: In the in vitro experiments, L929 mouse broblasts and two tumor cell lines (squamous cell carcinoma (SCC VII) and fibrosarcoma (FsaR)) were used. The cells were cultured with 0.1 or 0.01 mg/ml TQ or THQ for 24 h, and cytotoxicity assay was performed with the use of crystal violet staining technique. For in vivo antitumor efficiency evaluation of new compounds two murine tumor models (fibrosarcoma (FsaR) and squamous cell carcinoma (SCC VII)) were used. The used dose was equal for both substances. Antitumor effect of 4 intratumoral injections of TQ and THQ at the dose of 5 mg/kg was evaluated by comparison of tumor growth kinetics between treated and control animals. RESULTS: In vitro study showed that TQ and THQ exhibit statistically significant cytotoxic activity (p less, similar 0.01). The cytotoxic activity was dose dependent and more expressed against tumor cells than against L929 fibroblasts. The result of antitumor activities of TQ and THQ in vivo reached TGI = 52% and it was statistically significant (p less, similar 0.05). CONCLUSION: The results indicate that THQ antitumor activity may be improved with further dose increase of the investigated substance.

Antitumor efficiency of novel fluoro-substituted 6-amino-2-phenylbenzothiazole hydrochloride salts in vitro and in vivo.

The purpose of this study was to investigate antitumor activity of novel fluoro-substituted 6-amino-2-phenylbenzothiazole hydrochloride salts in vitro and in vivo. A novel series of hydrochloride or dihydrochloride salts of the novel 2-(fluoro-substituted phenyl)-6-aminobenzothiazoles (5-7) have been prepared in multistep synthesis starting from 3- or 4-fluorobenzaldehydes and 2-amino-5-nitrothiophenol and evaluated for their antiproliferative activity against human cervical (HeLa), breast (MCF-7), colon (CaCO-2), and laryngeal (Hep-2) carcinomas and against fibroblast cell lines (WI-38). Also, antitumor activity of these compounds was evaluated in vitro and in vivo against murine melanoma (B16-F10), fibrosarcoma (FsaR), and squamous cell carcinoma (SCCVII). The tested compounds were found to exert good cytotoxic activity in vitro. The cytotoxic effect was selective, cell specific, and dose dependent, between 33 microM for MCF-7 and 110 microM for WI-38. Benzothiazoles reduced de novo protein and DNA synthesis up to 75%. All examined benzothiazoles had significant antitumor activity in vivo against melanoma B16-F10, fibrosarcoma, and squamous cell carcinoma. The best therapeutic results were achieved when therapy started 7 days after tumor cell implantation and when benzothiazoles were given repeatedly five times every 2 days, i.e., on day 7, 9, 11, 13, and 15 after transplantation of tumor cells.

Cell killing of melanoma B16 in vivo by hyperthermia and cytotoxins.

The aim of this study was to investigate antitumour activity of cisplatin, dacarbasine, cyclophosphamide and a new compound from the nitrosourea group--acetamido-CNU ((2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)-urea)--applied with or without local hyperthermia (43.5 degrees C/60 min). The tumour model for the investigation of antitumour activity was a mouse melanoma B16 transplanted into the footpad. Dacarbasine, cyclophosphamide and acetamido-CNU applied as a single treatment had statistically significant antitumour activity, while cisplatin applied as a single agent had no effect. Local hyperthermia alone had statistically significant antitumour activity. The best therapeutic effect (synergistic) was obtained when combined treatment (cytotoxins plus local hyperthermia) was used. Synergistic therapeutic results were achieved even when cisplatin and hyperthermia were combined, although cisplatin was ineffective when given as a single agent. Therapeutic results achieved with acetamido-CNU (newly synthesized compound) applied alone were similar to the therapeutic results achieved with dacarbasine or cyclophosphamide. In combined therapy (acetamido-CNU + HT), achieved therapeutic results were significantly better (p < 0.05) than results achieved by combining cisplatin and hyperthermia or dacarbasine and hyperthermia.

Immunostimulatory effect of natural clinoptilolite as a possible mechanism of its antimetastatic ability.

PURPOSE: Many biochemical processes are closely related to ion exchange, adsorption, and catalysis. Zeolites reversibly bind small molecules such as oxygen or nitric oxide; they possess size and shape selectivity, the possibility of metalloenzyme mimicry, and immunomodulatory activity. These properties make them interesting for pharmaceutical industry and medicine. METHODS: The experiments were performed on mice. Different biochemical and molecular methods were used. RESULTS: Micronized zeolite (MZ) administered by gastric intubation to mice injected with melanoma cells significantly reduced the number of melanoma metastases. In mice fed MZ for 28 days, concentration of lipid-bound sialic acid (LSA) in serum increased, but lipid peroxidation in liver decreased. The lymphocytes from lymph nodes of these mice provoked a significantly higher alogeneic graft-versus-host (GVH) reaction than cells of control mice. After i.p. application of MZ, the number of peritoneal macrophages, as well as their production of superoxide anion, increased. However, NO generation was totally abolished. At the same time, translocation of p65 (NFkappaB subunit) to the nucleus of splenic cells was observed. CONCLUSION: Here we report antimetastatic and immunostimulatory effect of MZ and we propose a possible mechanism of its action.

[Vitreoretinal surgery in war injuries of the eye]. / Vitreoretinska hirurgija kod ratnih povreda oka.

Results of vitreoretinal surgeries of 647 severe penetrating injuries of the eye, sustained during the war operations in the territory of former Yugoslavia, in the period from 01.07.1991. to 31.12.1998. were analyzed in the paper. A total of 558 pars plana vitrectomies, 459 intrabulbar foreign bodies' extractions and 360 surgeries of traumatic retinal detachments (89 in the conventional way) were performed at the Clinic of Ophthalmology--Department for Vitreoretinal Surgery of the Military Medical Academy. Certain innovations in the surgical treatment of the severely injured eye with the damage of posterior eye segment, as well as the lens, were presented. In those cases, (simultaneously with pars plana vitrectomy, and extraction of retained intrabulbar foreign body), the primary intraocular lens (IOL) implantation was also performed, particularly posterior chamber IOL implantation following lensectomy, as well as phacoemulsification.

Natural zeolite clinoptilolite: new adjuvant in anticancer therapy.

Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.

The effect of the zeolite clinoptilolite on serum chemistry and hematopoiesis in mice.

Zeolites are natural or synthetic crystalline alumosilicates with ion exchanging properties. Supplied in fodder, they promote biomass production and animal health. Our aim was to assess the effects of the natural zeolite, clinoptilolite, on hematopoiesis, serum electrolytes and essential biochemical indicators of kidney and liver function in mice. Two preparations differing in particle size were tested: a powderized form obtained by countercurrent mechanical treatment of the clinoptilolite (MTCp) and normally ground clinoptilolite (NGCp). Young adult mice were supplied with food containing 12.5, 25 or 50% clinoptilolite powder. Control animals received the same food ration without the clinoptilolite. After 10, 20, 30 and 40 days, six animals from each group were exsanguinated to obtain blood for hematological and serum for biochemical measurements as well as to collect femoral bone marrow for determination of hematopoietic activity. Clinoptilolite ingestion was well tolerated, as judged by comparable body masses of treated and control animals. A 20% increase of the potassium level was detected in mice receiving the zeolite-rich diet, without other changes in serum chemistry. Erythrocyte, hemoglobin and platelet levels in peripheral blood were not materially affected. NGCp caused leukocytosis, with concomitant decline of the GM-CFU content in the bone marrow, which was attributed to intestinal irritation by rough zeolite particles. The mechanically treated clinoptilolite preparation caused similar, albeit less pronounced, changes. In a limited experiment, mice having transplanted mammary carcinoma in the terminal stage showed increased potassium and decreased sodium and chloride levels, severe anemia and leukocytosis, decreased bone marrow cellularity and diminished content of hematopoietic progenitor cells in the marrow. The clinoptilolite preparations ameliorated the sodium and chloride decline, whereas the effects on hematopoiesis were erratic.

[Retinal detachment in the pseudophakic eye]. / Ablacija retine u pseudofaknom oku.

Retinal detachments in pseudophakic eyes and their diagnostic and therapeutic features are analyzed. The authors pointed out diagnostic problems in detection of changes on the fundus significant for the onset of retinal detachment and difficulties in the detection of retinal rupture as important elements for the successful outcome of surgical treatment. The most frequent intra and postoperative risk factors for the occurrence of retinal detachment in pseudophakic eyes are presented. We have analyzed 16 patients treated by conventional surgical procedures as well as by inner silicone oil tamponade following pars plana vitrectomy in the Clinic of Ophthalmology of the Military Medical Academy during 1999. Results of surgical treatment of retinal detachments in pseudophakic eyes are in correlation with treatment results presented by other authors.

Correlation of two methods for determination of cathepsin D in breast carcinoma (immunohistochemistry and ELISA in cytosol).

We have studied the correlation of two methods (immunohistology and ELISA in cytosol) of cathepsin D (CD) determination in breast carcinoma patients. Fifty six specimens of tumor tissue were collected consecutively, and CD expression in tumor tissue and tissue macrophages was determined by standard immunohistochemistry using the aNCL-CDm anti-cathepsin D mouse monoclonal antibody (Novocastra Laboratories Ltd., Newcastle, UK). Additionally, CD concentration was determined by ELISA in cytosol of the same breast carcinoma specimens. CD positivity was correlated with tumor size, histological grade of tumor, and the cytosol progesterone adn estrogen receptor concentrations. There was no statistically significant correlation between examined parameters and either CD positivity by immunohistochemistry or cytosol CD concentration. The correlation between CD expression in tumor cells of breast carcinoma by immunohistochemistry and cytosol CD positivity was not found either. However, there was a significant association between abundance of CD positive stromal macrophages and cytosol CD concentration in all histological tumor types (p < 0.05). CD positive macrophages were abundant in most of cytosol CD positive specimens. These results suggest that breast cancer cytosol CD concentration is the cumulative result of CD content in both carcinoma cells and stromal macrophages.

Histomorphologic and histochemical characteristics of carpal glands (Glandulae carpeae) in domestic swine (Sus scrofa domesticus) and wild swine (Sus scrofa ferus).

Investigations were carried out on samples from carpal glands of domestic and wild swine of different ages. The anatomic position of carpal glands is identical in domestic and wild swine. Glandular acini in domestic swine are bigger and more scarce than in wild swine. LDH activity is more intense in wild swine, while the activity of the investigated diaphorases is almost even. The activity alkaline and acid phosphatase is stronger in domestic swine, while the activity of non-specific esterases is present only in wild animals. The more or less manifest presence of all investigated enzymes indicates that carpal glands in domestic and wild porcine specimens are very active metabolically. Regarding the intensity of enzymatic reactions, some differences are probably due to different sexual maturity, as well as to the method of feeding and housing in these groups of animals.

Oestrogen and progesterone receptors in primary breast cancer: a population study.

The results of a single centre study on cytoplasmic oestrogen (ER) and progesterone (PR) receptors and their combinations in primary breast cancer of 1957 patients from various parts of Croatia are presented. The frequency of ER+ tumours and tumour mean ER concentration were higher in patients over 50 years of age, while PR frequency and concentration were similar in patients over and under 50. The ER concentration was positively correlated with the age of patients, but the age-related increase in ER concentration appeared between 50 and 70 years of age. The pattern of receptor coexistence was age related. The frequency of ER+PR+ and ER+PR- increased and that of ER-PR+ and ER-PR- tumours decreased with the age of patients. The concentrations of ER and PR were higher in ER+PR+ than in ER+PR- or ER-PR+ tumours, respectively. When the patients were divided into groups under and over 50 years of age these differences appeared only in the latter group, while in the former the concentrations of ER were similar in ER+PR+ and in ER+PR- tumours, and the concentration of PR was higher in ER+PR+ than in ER-PR+ tumours. These data suggest a biological difference between breast cancers with various receptor combinations, as well as a difference in pathogenesis of the receptor negative and positive breast cancer.

Sex steroid receptors in intracranial tumors.

Estrogen (ER), progestin (PGR), and androgen (AR) receptors were assayed in cytosols prepared from 38 various intracranial tumors. The receptors were in the following proportions (number of receptor-positive/number of tumors examined): meningiomas were positive for PGR (4/6) and AR (2/5); glioblastomas were also positive for PGR (3/21) and AR (7/21); astrocytomas were positive only for PGR (4/5); and oligodendrogliomas only for AR. In two hamartomas AR was present, while in one chordoma both PGR and AR were present. In this latter tumor ER were not assayed due to insufficient material. The receptors were present in concentrations between 10 and 20 fmol/mg protein. Exceptions were two meningiomas and a chordoma with a high concentration of PGR and AR. Our results support the notion that a proportion of intracranial tumors contains sex steroid receptors, and some of these tumors might be hormonally dependent.

Androgen receptors in normal and neoplastic laryngeal tissue.

Androgen cytoplasmic (ARc) and nuclear (ARn) receptors were analyzed in normal and cancerous laryngeal tissue obtained from male and female patients. In men, neither ARc nor ARn were detectable in most of the normal larynx tissue specimens, while either ARc, ARn, or both were present in eight of 16 laryngeal carcinomas. In women, all three normal and two of four malignant laryngeal tissue specimens possessed ARc without ARn. The presence of androgen receptors in some laryngeal carcinomas shows that these tumors are possibly hormone sensitive, and hormonal therapy should be considered in treating these tumors.

Lymphocyte reactivity to mitogens and tumor sex steroid receptors in breast cancer patients.

In 60 breast cancer patients in stages I and II the blastogenic transformation of peripheral blood lymphocytes after phytohaemagglutinin (PHA) pokeweed mitogen (PWM) and concanavalin A (Con A) stimulation were assayed and estrogen (ER) and progesterone (PgR) receptor concentrations in tumor cytosol were measured. A negative correlation between lymphocyte reactivity to the mitogens and tumor steroid receptors concentration was found. The lymphocyte response to the mitogens in the patients with ER-PgR-tumors (R-) was significantly higher than in those with tumors either ER+PgR-or ER-PgR+ (R+) or ER+PgR+ (R++). There was also a negative correlation between lymphocyte response to PHA and either ER or PgR concentrations in the tumors. These results suggest that the presence of steroid receptors in tumor cells may be associated with the depression of immunological reactivity in breast cancer patients.