Dosimetry for lung tumorigenesis induced by urethane, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[a]pyrene (B[a]P) in A/JJmsSlc mice.

Some chemicals are known to be lung carcinogens in rodents. While many studies using two-stage models have administered medium or high doses to mice, few have tested lower doses. The dose dependence of urethane, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and benzo[a]pyrene (B[a]P), three well-known lung carcinogens at high doses, has not been sufficiently reported in lower dose ranges. Our study evaluated the tumorigenicity of urethane, NNK, and B[a]P at 26 weeks after a single intraperitoneal administration of each compound within medium to low dose in male and/or female A/JJmsSlc (A/J) mice. Dose-dependent tumorigenesis was demonstrated histopathologically for the three compounds. These results suggested that the tumorigenicity of these chemicals is dose dependent in A/J mice, even at lower doses than previously reported.

Characterization of biochemical, functional and structural changes in mice respiratory organs chronically exposed to cigarette smoke.

Female C57BL/6 mice were exposed to mainstream cigarette smoke at 600 µg WTPM/L, 4 h/day and 5 days/week for up to 52 weeks. At 26, 52 and 65 weeks (52 weeks of exposure plus 13 weeks of no exposure), lungs were assessed for inflammation, function, histopathology and morphometry. Structural changes were observed and accompanied by altered lung function at 26 and 52 weeks (e.g. increase of static compliance and hysteresis, and decrease of elastance). Lung morphometry quantified significant increase in airspace enlargement at 52 weeks. Chronic smoke exposure induced inflammation in respiratory organs, e.g. mixed inflammatory cell infiltrates, perivascular lymphocyte infiltrates and pigmented alveolar macrophages in the lungs. Minimal or mild alveolar emphysema was diagnosed in 70% by 26 weeks or 80% by 52 weeks. After 13 weeks of recovery, most biochemical, histopathological and morphometrical alterations were restored, while emphysema was observed to persist at 18% incidence by 65 weeks. In conclusion, the employed exposure conditions induced emphysematous changes in the lungs, accompanied by altered lung function and morphological/histopathological changes. Following the 13 weeks of no exposure, morphological changes persisted, although some functional/biochemical alterations regressed.

Biological responses in rats exposed to mainstream smoke from a heated cigarette compared to a conventional reference cigarette.

The heated cigarette (HC) generates mainstream smoke by vaporizing the components of the tobacco rod using a carbon heat source at the cigarette tip. Mainstream smoke of HC contains markedly less chemical constituents compared to combusted cigarettes. Mainstream smoke from HC was generated under Health Canada Intense regimen and its biological effects were compared to those of Reference (3R4F) cigarettes, using nose-only 5-week and 13-week inhalation studies. In the 13-week study, SD rats were necropsied following exposure to mainstream smoke from each cigarette at 200, 600 or 1000 µg wet total particulate matter/L for 1 h/day, 7 days/week or following a 13-week recovery period. Histopathological changes in the respiratory tract were significantly lesser in HC groups; e.g. respiratory epithelial hyperplasia in the nasal cavity and accumulation of pigmented macrophages in alveoli. After a 13-week recovery, the lesions were completely or partially regressed, except for accumulation of pigmented macrophages in alveoli, in both HC and 3R4F groups. In the 5-week study, SD rats were necropsied following exposure to mainstream smoke of either cigarette at 600 or 1000 µg/L for 1 h, two times/day (with 30 min interval), 7 days/week or following a 4-week recovery period. Bronchoalveolar lavage fluid (BALF) analysis of neutrophil percentages and enzyme levels like γ-GT, ALP and LDH indicated that pulmonary inflammation was significantly less in HC groups compared to 3R4F groups. In conclusion, HC demonstrated significantly lower biological effects compared to 3R4F, based on the BALF parameters and histopathology.

Comparison of dermal tumor promotion activity of cigarette smoke condensate from prototype (heated) cigarette and reference (combusted) cigarette in SENCAR mice.

Test cigarette (prototype "heated" cigarette) was evaluated on its dermal tumor promotion activity in SENCAR mice relative to conventional 3R4F cigarette. Mainstream cigarette smoke was generated under the modified Health Canada Intensive Regimen, and smoke condensate (CSCs) were collected using cold traps and extracted with acetone. Female mice received a topical application of 7,12-dimehtylbenz(a)anthracene (DMBA) as the tumor initiator on the back skin during Week 1. Subsequently, CSC was repeatedly applied as the tumor promoter at 5 doses, up to 30 mg tar/application, three times per week for 30 weeks. Test groups showed a clearly longer latency at lower doses (⩽15 mg), but the difference was less clear at higher doses (⩾22.5 mg), while mortalities were not affected throughout the study. Test groups also had consistently lower incidence and multiplicity of neoplasms, as well as lower incidences of non-neoplastic changes (e.g., inflammations and squamous epithelial hyperplasia on the site of application). The group without DMBA initiation did not induce any neoplasm but the respective Reference group showed an increase in tumorigenicity. In conclusion, the study demonstrated significant reduction in dermal irritancy and tumorigenicity of Test CSC compared to Reference CSC.

Comparison of biological responses in rats under various cigarette smoke exposure conditions.

A variety of exposure regimens of cigarette smoke have been used in animal models of lung diseases. In this study, we compared biological responses of smoke exposure in rats, using different smoke concentrations (wet total particulate matter [WTPM]), daily exposure durations, and total days of exposure. As a range-finding acute study, we first compared pulmonary responses between SD and F344 strains after a single nose-only exposure to mainstream cigarette smoke or LPS. Secondly, F344 rats were exposed to cigarette smoke for 2 or 13 weeks under the comparable daily exposure dose (WTPM concentration x daily exposure duration; according to Haber's rule) but at a different WTPM concentration or daily exposure duration. Blood carboxylhemoglobin was increased linearly to the WTPM concentration, while urinary nicotine plus cotinine value was higher for the longer daily exposure than the corresponding shorter exposure groups. Gamma glutamyl transferase activity in bronchoalveolar lavage fluid (BALF) was increased dose dependently after 2 and 13 weeks of cigarette smoke exposure, while the neutrophil content in BALF was not increased notably. Smoke-exposed groups showed reduced body weight gain and increased relative lung and heart weights. While BALF parameters and the relative lung weights suggest pulmonary responses, histopathological examination showed epithelial lesions mainly in the upper respiratory organs (nose and larynx). Collectively, the results indicate that, under the employed study design, the equivalent daily exposure dose (exposure concentration x duration) induces equivalent pulmonary responses in rats.

Mechanisms of olive leaf extract-ameliorated rat arthritis caused by kaolin and carrageenan.

Olive leaf extract (OLE) has antioxidant and antiinflammatory actions. However, the role of OLE in mechanical inflammatory arthritis (osteoarthritis, OA) is unclear. This study investigated the effect of OLE on the development of kaolin and carrageenan-induced arthritis, a murine model of OA. Administration of OLE significantly ameliorated paw swelling, the paw Evans blue content and the histopathological scores. In the human monocyte cell line, THP-1, the OLE reduced the LPS-induced TNF-α production and was dose dependent. Croton oil-induced ear edema in mice also revealed that treatment with OLE suppressed ear edema, myeloperoxidase (MPO) production and was dose dependent. These results indicated that OLE is an effective antiarthritis agent through an antiinflammation mechanism. Also OLE may be beneficial for the treatment of OA in humans.

Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies.

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1 h or 4 h/day) at equivalent daily exposure amount (concentration × time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1 h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.

Identification of antidepressant-like ingredients in ginseng root (Panax ginseng C.A. Meyer) using a menopausal depressive-like state in female mice: participation of 5-HT2A receptors.

RATIONALE: After reports of adverse effects with hormone replacement therapy, such as reproductive and breast cancer and coronary heart disease, much attention has been given to the development of new remedies to alleviate menopausal depression in women, but methods for their preclinical evaluation have not been clarified. We previously developed a procedure to predict the drug effect on the menopausal depressive-like state in female mice. OBJECTIVES: We attempted to identify psychoactive components from ginseng root, one of the earliest known materials for menopausal disorder, and to clarify the possible mechanism involved. METHODS: As an index of a depressive-like state, we used the prolongation of immobility time induced by an ovariectomy during the forced swimming test. Chronic treatment with the candidate substance began the day after ovariectomy and continued for 14 days. To examine whether the 5-HT(2A) receptor antagonist ritanserin antagonized the antidepressant-like effect of ginsenoside Rb(1), ritanserin was given as pretreatment 15 min before the daily administration of ginsenoside Rb(1) and the antagonistic effect was compared with ginsenoside Rb(1) alone. RESULTS: Ginsenoside Rb(1) and compound K were active ingredients that dose-dependently prevented the prolongation of immobility time induced by ovariectomy. Co-administration of ritanserin, a 5-HT(2A)-receptor antagonist, antagonized the effect of ginsenoside Rb(1). CONCLUSIONS: We suggest that ginsenoside Rb(1) and its metabolite, compound K, are antidepressant-like components of the ginseng root, and that 5-HT(2A) receptors may play an important role in mediating the antidepressant-like effect of ginsenoside Rb(1).

Comparison of the physiological and morphological effects of cigarette smoke exposure at comparable weekly doses on Sprague-Dawley rats.

A variety of dose x duration exposure regimens have been used in inhalation toxicity studies using rodents. We evaluated the effects of differences in smoke concentration and daily exposure duration under similar weekly cumulative exposures in rats to determine potential variation in type and severity of adverse effects in 13-week exposure studies. The weekly cumulative dosages were 2100 and 4200 µg wet total particle matter (WTPM)/L, and the daily exposure durations were 1 and 6 h. Weekly exposure duration was 5 or 7 days/week for groups exposed 1 h/day and 7 days/week for groups exposed 6 h/day. Recovery duration was 6 and 13 weeks. Mainstream smoke exposure suppressed body weight (BW) gain in both regimens. Lower dose groups exposed 1 h/day had a consistently greater of BW gain compared with corresponding 6 h/day groups. Respiratory rate, tidal volume, and minute volume (MV) were suppressed in a dose-dependent manner in both regimens. Higher MV in rats exposed for 6 h/day compared with rats exposed 1 h/day suggested that a lower concentration for longer duration resulted in a greater total inhaled mass (TIM) in rats exposed 6 h/day. Groups exposed for 6 h/day had lower blood carboxyhemoglobin and plasma nicotine levels than groups exposed 1 h/day, reflecting the lower carbon monoxide (CO) and WTPM concentrations in the 6 h/day groups. Data from examination of bronchoalveolar lavage fluids (BALF) and respiratory tract tissues indicated comparable effects between both regimens. Exposure-induced histopathological changes regressed similarly for both regimens after the recovery periods.

Mechanism of the antiviral effect of hydroxytyrosol on influenza virus appears to involve morphological change of the virus.

Hydroxytyrosol (HT), a small-molecule phenolic compound, inactivated influenza A viruses including H1N1, H3N2, H5N1, and H9N2 subtypes. HT also inactivated Newcastle disease virus but not bovine rotavirus, and fowl adenovirus, suggesting that the mechanism of the antiviral effect of HT might require the presence of a viral envelope. Pretreatment of MDCK cells with HT did not affect the propagation of H9N2 virus subsequently inoculated onto the cells, implying that HT targets the virus but not the host cell. H9N2 virus inactivated with HT retained unaltered hemagglutinating activity and bound to MDCK cells in a manner similar to untreated virus. Neuraminidase activity in the HT-treated virus also remained unchanged. However, in the cells inoculated with HT-inactivated H9N2 virus, neither viral mRNA nor viral protein was detected. Electron microscopic analysis revealed morphological abnormalities in the HT-treated H9N2 virus. Most structures found in the HT-treated virus were atypical of influenza virions, and localization of hemagglutinin was not necessarily confined on the virion surface. These observations suggest that the structure of H9N2 virus could be disrupted by HT.

Effects of hydroxytyrosol-20 on carrageenan-induced acute inflammation and hyperalgesia in rats.

Hydroxytyrosol (HT) is a simple phenol compound extracted from olive leaves. The content of HT in the studied preparation was about 20%, and the preparation was called hydroxytyrosol-20 (HT-20). HT has antioxidant and antiinflammatory activities. There has been no report so far on the efficacy of HT-20 in carrageenan-induced acute inflammation and hyperalgesia in rats. Therefore, the aim of this study was to assess the inhibitory role of HT-20 on carrageenan-induced swelling and hyperalgesia of rat paw. Paw inflammation was assessed by the increase in paw volume and hyperalgesia. The rat paws were cut out under ether anesthesia at 270 min after administration of carrageenan. The tissue of the right paw was isolated separately from the individual rat. The levels of the tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 10 (IL-10) mRNA in the tissue were estimated by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the paw pressure thresholds of rats orally administered HT-20 significantly increased at 210, 240 and 270 min after administration of carrageenan, compared with corresponding basal paw pressure thresholds; the degree of swelling of the right hind paw showed a statistically significant reduction, compared with rats in the carrageenan-treated control. In this model, HT-20 appears to decrease pro-inflammatory cytokines IL-1beta and TNF-alpha and not to increase the antiinflammatory cytokine mRNA expression of IL-10.

Inhibition of acrylamide genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol.

The chemoprotective effect of hydroxytyrosol (HT), a strong antioxidant compound from extra virgin olive oil, against acrylamide (AA)-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The micronucleus test (MNT) assay was used to monitor genotoxicity. In MNT, we found that HT at all tested concentrations (12.5-50 microM) significantly reduced the micronuclei frequencies in a concentration-dependent manner caused by AA. In order to clarify the underlying mechanisms we measured the intracellular reactive oxygen species (ROS) formation using 2,7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The rate-limiting enzyme in GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS) and gamma-GCS was measured using Western blotting. The results showed that HT significantly concentration-dependent reduced the genotoxicity caused by AA. Furthermore, HT was able to reduce intracellular ROS formation and attenuate GSH depletion caused by AA in a concentration-dependent manner. It was also found that HT enhanced the expression of gamma-GCS in HepG2 cells treated with 10 mM AA using immunoblotting in a concentration-dependent manner. The results showed that HT reduced the AA-induced genotoxicity by decreasing the ROS level and increasing the GSH level. The data strongly suggest that HT have significant protective ability against AA-induced genotoxicity in vitro.

The anti-atherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis.

BACKGROUND: The anti-atherogenic effect of olive leaf extract is supposed to be related to its activities of anti-oxidation and anti-inflammation. AIM OF THE STUDY: To prove the effect of anti-atherosclerosis by olive leaf extract (OLE) and to elucidate the mechanism behind. METHODS: Twenty-four rabbits were assigned to the control, high lipid diet (HLD) and OLE group that were fed with standard diet, HLD and HLD supplemented with OLE, respectively. Serum levels of atherosclerosis related markers, triglyceride (TG), total cholesterol (T-CHO), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and malondialdehyde (MDA) were detected at the ends of week 2, 4 and 6. Surface lesions and thickness of intimas were measured at the end of week6. The protein and/or mRNA expressions of inflammation factors, monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor alpha (TNF-alpha) were investigated by immunohistochemistry and RT-PCR. RESULTS: Atherosclerotic lesions were found in the HLD and OLE groups but not in the control group. In comparison with that in the HLD group, reduced size and thickness of intima (0.31 +/- 0.26 in the HLD group versus 0.10 +/- 0.03 mm in the OLE group) were found in the OLE group. The MDA level, an indicator of antioxidant status, was 35.27 +/- 15.37 in the HLD group and 20.63 +/- 11.52 nmol/ml in the OLE group. The level of CHO, TG and LDL-C were 104.46 +/- 30.34, 2.48 +/- 1.11, 82.83 +/- 28.44 mmol/l in the HLD group versus 83.03 +/- 27.23, 1.84 +/- 0.44, 59.51 +/- 23.72 mmol/l in the OLE group. Down-regulated expressions of MCP-1, VCAM-1, NF-kappaB and TNF-alpha at both protein and mRNA level (P < 0.05) were also found with the administration of OLE. CONCLUSION: This study proved the effect of OLE on inhibition of atherosclerosis, which is related to the suppressed inflammatory response.

Inhibition of Sudan I genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol.

The chemoprotective effect of hydroxytyrosol (HT) against Sudan I-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The comet assay and micronucleus (MN) assay were used to monitor genotoxicity. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). The levels of oxidative DNA damage and lipid peroxidation were estimated by immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), respectively. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The results showed that HT significantly reduced the genotoxicity caused by Sudan I. Furthermore, HT ameliorated lipid pexidation as demonstrated by a reduction in TBARS formation and attenuated GSH depletion in a concentration-dependent manner. It was also found that HT reduced intracellular ROS formation and 8-OHdG level caused by Sudan I. These results strongly suggest that HT has significant protective ability against Sudan I-induced genotoxicity.

Intestinal epithelial cells absorb gamma-tocotrienol faster than alpha-tocopherol.

To elucidate the transepithelial transport characteristics of lipophilic compounds, the cellular uptake of tocopherol and tocotrienol isomers were investigated in Caco2 cell monolayer models. These vitamin E isomers formed mixed micelles consisting of bile salts, lysophospholipids, free fatty acid, and 2-monoacylglycerols, then the micelles were supplied to Caco2 cells. The initial accumulation of tocotrienol isomers in Caco2 cells was larger than those of corresponding tocopherol isomers. There was little difference among the cellular accumulations of four tocopherol isomers. These findings suggested that the difference between the molecular structures of the C16 hydrocarbon chain tail in tocopherol and tocotrienol was strongly responsible for the rapid epithelial transport into the Caco2 cells membranes rather than the difference in the molecular structures of their chromanol head groups. Furthermore, the secretion of alpha-tocopherol and gamma-tocotrienol from Caco2 cells was investigated using Caco2 cells plated on a transwell. The time courses of their secretions from Caco2 cells showed that the initial secretion rate of gamma-tocotrienol was also larger than that of alpha-tocopherol. To investigate the intestinal uptake of alpha-tocopherol and gamma-tocotrienol in vivo, the mice were fed single doses of alpha-tocopherol or gamma-tocotrienol with triolein. The gamma-tocotrienol responded faster in plasma than alpha-tocopherol, although the maximal level of gamma-tocotrienol was lower than that of alpha-tocopherol. This suggested that the intestinal uptake properties of administered alpha-tocopherol and gamma-tocotrienol would characterize their plasma level transitions in mice.

Effects of flavoring and casing ingredients on the toxicity of mainstream cigarette smoke in rats.

A series of in vitro and in vivo studies evaluated the potential effects of tobacco flavoring and casing ingredients. Study 1 utilized as a reference control cigarette a typical commercial tobacco blend without flavoring ingredients, and a test cigarette containing a mixture of 165 low-use flavoring ingredients. Study 2 utilized the same reference control cigarette as used in study 1 and a test cigarette containing eight high-use ingredients. The in vitro Ames Salmonella typhimurium assay did not show any increase in mutagenicity of smoke condensate from test cigarettes designed for studies 1 and 2 as compared to the reference. Sprague-Dawley rats were exposed by nose-only inhalation for 1 h/day, 5 days/wk for 13 wk to smoke from the test or reference cigarettes already described, or to air only, and necropsied after 13 wk of exposure or following 13 wk of recovery from smoke exposure. Exposure to smoke from reference or test cigarettes in both studies induced increases in blood carboxyhemoglobin ((COHb)) and plasma nicotine, decreases in minute volume, differences in body or organ weights compared to air controls, and a concentration-related hyperplasia, squamous metaplasia, and inflammation in the respiratory tract. All these effects were greatly decreased or absent following the recovery period. Comparison of rats exposed to similar concentrations of test and reference cigarette smoke indicated no difference at any concentration. In summary, the results did not indicate any consistent differences in toxicologic effects between smoke from cigarettes containing the flavoring or casing ingredients and reference cigarettes.

Factors producing a menopausal depressive-like state in mice following ovariectomy.

RATIONALE: Bilateral ovariectomy in female mice produces a menopausal depressive-like state but the factors responsible for the phenomenon are unknown. OBJECTIVES: We elucidated methodological issues related to establishing this mouse model and investigated a possible mechanism underlying the depressive-like state of ovariectomized mice. METHODS: We removed both ovaries of female ICR mice at 9 weeks of age. Changes in the immobility time during the forced swimming test as a function of the time interval between ovariectomy and behavioral testing were determined on nine different days after surgery. To assess behavioral specificity, the elevated plus-maze (EPM) behavior and spontaneous activity were measured. With respect to the effect of ovariectomy on the immobility time, we compared ICR mice with three other strains of mice (C57BL/6J, DBA/2N, and CD-1). Finally, we investigated the effects of (-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and (+/-)-8-hydroxy-2-(N,N-di-n-propylamino) tetralin (8-OH-DPAT) on the immobility time of ovariectomized mice. RESULTS: A significant effect on the prolongation of immobility was observed between 12 and 18 days after ovariectomy. Ovariectomy did not alter either the EPM behavior or spontaneous activity. Of the four strains of mice, only DBA mice did not show any significant prolongation of immobility after ovariectomy. Acute or chronic treatment with DOI (0.5 or 1.0 mg kg(-1)) significantly prevented the prolongation of immobility time, whereas acute and chronic treatments with 8-OH-DPAT (0.05, 0.5, or 1.0 mg kg(-1)) were ineffective. CONCLUSION: The present findings have potentially important implications for evaluating a candidate substance for the management of mood disorders in menopausal women.

Heme oxygenase-1 gene expression in human alveolar epithelial cells (A549) following exposure to whole cigarette smoke on a direct in vitro exposure system.

Many in vitro studies have employed cigarette smoke condensates or soluble smoke components to investigate the biological effects of cigarette smoke. However, neither of these methods evaluates the biological effects of fresh whole cigarette smoke. It is most desirable to conduct in vitro biological studies under conditions which accommodate the dynamic physicochemical character of fresh cigarette smoke. Previously we reported the development of a whole smoke exposure system to assess the biological effects of mainstream cigarette smoke. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX cultivation technique, which includes a systemized air/liquid interface methodology and exposes the cells to fresh smoke at every puff. The aim of this study was to adopt the other biological endpoint to our whole smoke exposure system. We focused on heme oxygenase (HO)-1 mRNA gene expression, an enzyme which has recently been shown to be highly responsible for oxidative stress. In the present study, a dose-response relationship between the HO-1 mRNA expression based on the reverse transcription real-time PCR method and total exposure to cigarette smoke was observed. When a Cambridge filter pad was placed between the cigarette and exposure module, to ensure the cells were only exposed to the gas/vapor phase, the latter, as well as the whole smoke, induced HO-1 mRNA dose dependently. For the next step, acetate plain and charcoal filters with the same pressure drop were prepared to assess the potential ability of charcoal filters with regard to the vapor phase performance. The results revealed reduced HO-1 mRNA gene expression when a charcoal filter was used. Direct whole smoke exposure is a significant approach and may reflect the conditions of exposure essentially resulting from direct contact between cells and a dynamic mixture of gaseous and particulate constituents. We were able to adopt a gene expression assay for oxidative stress to the whole smoke exposure system, following the adaptation of cytotoxicity assays. This system, which includes several advantages involving the post-exposure washing of cells, by adding the exchanging medium and assuring the exposure of the particulate phase through the sedimentation method, may have potential for further investigations into the molecular basis of smoking-related lung disease.

Animal model of menopausal depressive-like state in female mice: prolongation of immobility time in the forced swimming test following ovariectomy.

RATIONALE: The onset of menopause produces a depressive state in women, but the mechanism involved in menopause-induced depression is poorly understood. OBJECTIVES: Building upon previous studies that used the duration of immobility in male rodents during the forced swimming test as a behavioral measure of the depression-like state, we investigated whether the duration of immobility in female mice was altered following bilateral ovariectomy. We also evaluated the chronic effects of estradiol, antidepressants, scopolamine, and diazepam on both the duration of immobility and uterine weight. METHODS: We bilaterally resected the ovaries of ICR albino female mice at 9 weeks of age. The day after the surgery, drug treatment was started once per day for 2 weeks, after which, the behavioral test was administered. The duration of immobility was measured during the last 4 min of the 6-min trial. RESULTS: Bilateral ovariectomy significantly increased the duration of immobility. Chronic treatment with estradiol (15-30 microg kg(-1) day(-1)) prevented the prolongation of immobility following ovariectomy and produced a significant increase in uterine weight. Chronic treatment with imipramine, fluvoxamine, and milnacipran at 5, 10, and 15 mg kg(-1) day(-1), respectively, significantly reduced the duration of immobility, whereas treatment with scopolamine or diazepam (0.5, 1.0, and 2.0 mg kg(-1) day(-1) for both) was ineffective. CONCLUSION: Based on these results, we suggest that the prolongation of immobility in female mice following ovariectomy may be a useful tool for investigating menopausal depression.

Relationship between damaged fraction and reflected spectra of denaturing tissues.

BACKGROUND AND OBJECTIVES: During thermal therapy of tissue, such as induced by microwave heating, the initiation of denaturation should be monitored for proper thermal dosage. Additionally, denaturation should be confined to the pathologic volume, while preserving surrounding healthy tissue. The relationship between the damaged fraction and reflected spectra of denaturing tissues was investigated for a variation of the temperature of the tissues. STUDY DESIGN/MATERIALS AND METHODS: Denaturation of muscle, liver, and milk was studied in vitro by measuring the temperature-varying reflectance spectrum as heating occurs. A high-resolution fiber optic spectrometer was used to measure the reflectance changes. Temperature was monitored using a thermocouple embedded within the tissue along the side of the optical fiber probe. RESULTS: The values of average free energy to initiate denaturation in muscle and liver at about 60 degrees C were 94.8 and 96.3 kJ/mole, respectively. The reflectance spectra increased in amplitude for muscle and liver, and the peak shifted from approximately 700 to 720 nm in accordance with the damage fraction of tissue. The reflectance spectrum for milk was essentially unchanged. CONCLUSIONS: Spectral changes from heated muscle and liver reflect denaturation of proteins contained therein. The spectral information at 800 nm can be used to determine the average free energy for the initiation of denaturation.