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BACKGROUND AND PURPOSE So far, there is only limited information about the regulation of the endogenous synthesis of hydrogen sulfide (H2S), an important gaseous signalling molecule. This study was done to evaluate the redox-dependent signalling events that regulate the expression of the H2S synthesising enzyme cystathionine-γ-lyase (CSE) in rat mesangial cells. EXPERIMENTAL APPROACH The effects of platelet-derived growth factor (PDGF)-BB and antioxidants on CSE expression and activity in cultured rat renal mesangial cells were assessed. Activity of nuclear factor erythroid-2-related factor-2 (Nrf2) was measured as the binding capacity to a radiolabelled consensus element by electrophoretic mobility shift assay (EMSA). Furthermore, CSE and Nrf2 expression was analysed in a rat model of anti-Thy-1-induced glomerulonephritis by immunohistochemistry. KEY RESULTS Treatment of mesangial cells with PDGF-BB resulted in a marked time- and dose-dependent up-regulation of CSE mRNA and protein levels, as well as CSE activity accompanied with increased formation of reactive oxygen species. Remarkably, co-administration of antioxidants, such as N-acetylcysteine, ebselen or diphenylene iodonium chloride, drastically reduced PDGF-BB-induced CSE expression. PDGF-BB induced binding of Nrf2 to a corresponding consensus antioxidant element in a redox-dependent manner. Furthermore, PDGF-BB-induced CSE expression in mouse mesangial cells was completely abolished in Nrf2 knockout mice compared with wild-type mice. In a rat model of anti-Thy-1-induced proliferative glomerulonephritis, we observed a marked up-regulation of CSE protein paralleled by a stabilization of Nrf2 protein. CONCLUSIONS AND IMPLICATIONS PDGF-BB regulated CSE via a redox-mediated activation of Nrf2. Such action would aid the resolution of glomerular inflammatory diseases. LINKED ARTICLE This article is commented on by Gallyas, pp. 2228–2230 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01976.x

Objectives Anthracyclines are broad spectrum anticancer drugs with dose-dependent cardiotoxicity. Protein malnutrition commonly occurs in cancer patients and is considered a risk factor for development of cardiotoxicity. This study was designed to assess the modulatory effect of protein malnutrition on the pharmacokinetics and drug disposition properties of a single dose of doxorubicin and epirubicin and how these possible changes will affect the degree of cardiotoxicity of these drugs. Methods A single interperitoneal dose of 15 mg/kg of either doxorubicin or epirubicin was injected into rats fed with either normal protein diet or low-protein diet. The plasma concentration–time profiles of doxorubicin and epirubicin and their concentrations in different tissues were determined. Serum creatine kinase level was determined at different time intervals and histopathological examination of heart tissue was carried out. Key findings Protein malnutrition significantly altered the pharmacokinetics of doxorubicin and epirubicin, with a significant decrease in their elimination, and prolonged the exposure of the heart to these drugs. Histopathological examination and serum creatine kinase measurements supported the role of protein malnutrition in enhancement of anthracycline cardiotoxicity. Conclusions If similar alteration in anthracyclines' pharmacokinetics occurs in malnourished cancer patients, protein malnutrition will be a risk factor for development of anthracycline cardiotoxicity and dose adjustment will be required in nutritionally deprived patients.

We have addressed in this study the possible protective role of the main principle of turmeric pigment; curcumin on a murine model of ulcerative colitis (UC). Colitis was induced by administration of dextran sulfate sodium (DSS) (3% W/V) in drinking water to male Swiss albino rats for 5 consecutive days. DSS challenge induced UC model that was well characterized morphologically and biochemically. DSS produced shrinkage of colon length and increased the relative colon weight/length ratio accompanied by mucosal edema and bloody stool. Histologically, DSS produced submucosal erosions, ulceration, inflammatory cell infiltration and crypt abscess as well as epithelioglandular hyperplasia. The model was confirmed biochemically, and the test battery entailed elevated serum tumor necrosis factor (TNF-alpha) and colonic activity of myleoperoxidase (MPO). Colonic glutathione-S-transferase (GST) activity and its substrate concentration; GSH, were notably reduced, while lipid peroxidation, expressed as malondialdehyde (MDA) level, and total nitric oxide (NO) were significantly increased. Prior administration of curcumin (100mg/kg, IP) for 7 consecutive days ahead of DSS challenge mitigated the injurious effects of DSS and ameliorated all the altered biochemical parameters. These results suggest that curcumin could possibly have a protective role in ulcerative colitis probably via regulation of oxidant/anti-oxidant balance and modulation of the release of some inflammatory endocoids, namely TNF-alpha and NO.

Background/Aims: The objective of this study was to assess in vivo gene therapy of uterine leiomyomas in the Eker rat model using adenovirus (Ad)-mediated delivery of herpes simplex virus 1 thymidine kinase gene (HSV1TK) followed by ganciclovir (GCV) treatment. Methods: We randomized 27 female Eker rats with MRI-confirmed uterine leiomyomas to a single treatment with direct intra-tumor injection of Ad-HSV1TK/GCV, Ad-LacZ/GCV, or medium alone. Samples were collected from tumors, other body organs, and blood at 10, 20, and 30 days after treatment to assess the safety and efficacy of the treatment. Results: Ad-HSV1TK/GCV treatment significantly decreased uterine fibroid volume by 75 ± 16, 58.7 ± 6.3, and 67.5 ± 27.5%, of the pretreatment volume at days 10, 20, and 30, respectively. Ad-HSV1TK/GCV increased caspase-3 activity, Bax expression, and TUNEL apoptosis marker, and it decreased cyclin D1, PCNA, Bcl2, and PARP protein expressions. Ad transfection induced local CD4+ and CD8+ infiltration and serum anti-Ad antibodies. Additionally, Ad transfection was tumor-localized and safe to non-target tissues. Conclusion: These studies demonstrate a marked efficiency and high safety for the Ad-HSV1TK/GCV therapeutic approach in the context of Eker rat uterine leiomyomas and provide essential preclinical data for the development of Ad-HSV1TK/GCV gene therapy for uterine fibroids.

Context: Human uterine leiomyomas are very common smooth muscle cell tumors that occur in reproductive-age women and are the leading reason for performing hysterectomies. The present study was conducted to explore the potential mechanism behind the effects exerted by dominant-negative estrogen receptors (DNERs) delivered by adenovirus to leiomyoma cells to ascertain the utility of DNERs as a novel strategy for treatment of uterine fibroids. Objective and Methods: We investigated the ability of DNER to affect estrogen response element (ERE) activity induced by wild-type estrogen receptor (ER) by using the adenovirus ERE luciferase (AdERE-luc) system in ELT3 cells and the effect of graded doses of DNER (10, 50, and 100 plaque-forming units/cell) on the expression of some selected genes controlling cultured human leiomyoma cell proliferation (cyclin D1, Cox2, PCNA, VEGF, and EGF), apoptosis (Bcl2 and Bax), estrogen metabolism (COMT), and extracellular matrix formation(MMP1)aswellasprogesteronereceptors(AandB)wereassessed usingWesternblotanalysis.Thesegenesareallregulatedbyestrogen and/or progesterone. Results: DNER has the ability to suppress the ERE luc activity induced by wild-type ER (P 0.01) and significantly (P 0.05) reverse the expression of all estrogen- and progesterone-regulated genes in this study. Conclusions:These results suggest that interruption of the estrogen signaling pathway using DNER results in modulation of both estrogen- and progesterone-regulated genes that control leiomyoma cell apoptosis, proliferation, extracellular matrix formation, progesterone receptors, and estrogen metabolism, which might account for the DNER mechanism of action. (J Clin Endocrinol Metab 92: 3949–3957, 2007)

1. Doxorubicin (DOX), a standard chemotherapeutic anthracycline agent, causes a positive inotropic effect in guinea-pig isolated atria in a concentration-dependent manner with an ED(50) of 3.6 micromol/L. This increase in contractility is strictly related to the generation of reactive oxygen species (ROS) as a consequence of quinone metabolism. The ED(50) of DOX is significantly increased (P < 0.05) in the presence of 150 U superoxide dismutase (SOD). In the heart, DOX may be subjected to one- or two-electron reductions catalysed by flavoenzymes in the presence of suitable electron donors. Two-electron reduction is catalysed by NAD(P)H quinone acceptor oxidoreductase (DT-diaphorase; DTD). Whether DOX will be activated or detoxified by two-electron reduction is important for the understanding of the mechanism of both the toxic and antitumour actions of DOX. 2. In order to assess the role of DTD in cardiac responses to DOX, we examined the effect of both a specific inhibitor (dicoumarol) and an inducer (3-methylcholanthrene; MCA) of the enzyme on the inotropic action of DOX. 3. In guinea-pig isolated left atria, 4 micromol/L dicoumarol significantly enhanced the positive inotropic effect of DOX, especially at lower concentrations of DOX. In atria isolated from guinea-pigs treated with MCA (44 mg/kg, i.p. for 4 days), DTD activity was enhanced (approximately twice that of the control; P < 0.01), whereas the activity of glutathione S-transferase (GST) was not significantly altered. In these preparations, DOX caused a significantly lower increase in force of contraction than in atria isolated from untreated animals. 4. These results demonstrate that cardiac DTD does not contribute to ROS generation, but represents a detoxification system.

The effects of three natural phenolic acids (caffeic, ferulic, and p-coumaric) on the rat thyroid gland were examined in a 3-week oral-treatment study. Forty male Wistar albino rats, divided into groups of 10 rats each and fed iodine-rich diet, were administered by gastrointestinal tube saline (control), caffeic acid, ferulic acid, or p-coumaric acid at a dose level of 0.25 micromol/kg/day for 3 weeks. The mean absolute and relative thyroid weights in caffeic, ferulic, or p-coumaric acid groups were significantly increased to 127 and 132%, 146 and 153%, or 189 and 201% compared to control value, respectively. Histological examination of the thyroids of p-coumaric acid group revealed marked hypertrophy and/or hyperplasia of the follicles. Caffeic or ferulic groups showed slight to moderate thyroid gland enlargement. Thyroid lesions in p-coumaric acid group were associated with significant increases in cellular proliferation as indicated by [(3)H]thymidine incorporation. In addition, the goitrogenic effect of p-coumaric acid was further confirmed by significant decreases (50%) in serum tri-iodothyronine (T(3)) and thyroxine (T(4)), and a parallel increase (90%) in serum thyroid stimulating hormone (TSH) compared to control group. These results indicate that administration of p-coumaric acid at relatively high doses induces goiter in rats.

In the current study, we have investigated the bioeffects of repeated exposure to low-frequency (50 Hz) high-intensity (20 mT; 200 G) electromagnetic field (EMF) on some immune parameters in mice. The animals were exposed to EMF daily for 30 min three times per week for 2 weeks. We also studied the possible immunomodulatory effects of two anti-radical substances known to have non-specific immunostimulant effects namely, l -carnitine (200 mg/kg body weight i.p.) and Q10 (200 mg/kg body weight, p.o.). Both drugs were given 1 h prior to each EMF exposure. Immune endpoints included total body weight, spleen/body weight ratio, splenocytes viability, total and differential white blood cell (WBCs; lymphocytes, monocytes, neutrophils) counts, as well as the lymphocyte proliferation induced by the mitogens; phytohaemagglutinin (PHA), concanavalin-A (Con-A) and lipoploysaccharide (LPS). Magnetic field decreased splenocyte viability, WBCs count, as well as mitogens-induced lymphocyte proliferation. l -carnitine, but not Q10 could ameliorate the adverse effects of EMF on the vast majority of the immune parameters tested, suggesting a possible immunoprotective role of l -carnitine under the current experimental conditions.

The present study was designed to investigate the possible modulator effect of melatonin on uterine estrogen and progesterone receptors in rats as well as the uterine response to oxytocin. Non-pregnant rats were pretreated with melatonin in a dose of 0.8 mg kg(-1) per day for 15 consecutive days. Control animals received the vehicle. The uterus was dissected out and uterine contraction in one horne was recorded in vitro for each animal as a response to oxytocin (0.5 x 10(-11) to 2 x 10(-11)M). The other uterine horne was subjected to estrogen and progesterone receptors detection by immunohistochemical and image analysis techniques. The results reveal a significant reduction (59%) in the number of uterine estrogen receptors with concomitant increase in the progesterone receptors (53%) in melatonin-pretreated rats as compared to the control ones. In addition, our data show an inhibitory effect of melatonin on the uterine contraction as a response to oxytocin (0.5 x 10(-11), 1 x 10(-11), and 2 x 10(-11)M) amounting to 48, 77, and 59.5% reduction, respectively, in the amplitude of contraction as well as 62, 19.9, and 47% reduction in the area under the curve (AUC) of uterine contractions, respectively. The data, so far obtained, may indicate a possible relationship of melatonin-induced modulation of the number of estrogen and progesterone receptors and its inhibitory effect on uterine contraction. These findings merit further investigations on the possible beneficial role of melatonin in a plethora of hormone-dependent uterine disorders.

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Understanding the mechanisms involved with genetic susceptibility to environmental disease is of major interest to the scientific community. We have conducted an in vitro study to elucidate the involvement of polymorphic metabolizing genes on the genotoxicity of benzo[a]pyrene (BP). Blood samples from 38 donors were treated with BP and the induction of sister chromatid exchanges (SCE) and chromosome aberrations (CA) were evaluated. The latter is based on the tandem-probe fluorescence in situ hybridization (FISH) assay. The data indicate that the induction of genotoxicity was clearly determined by the inherited variant genotypes for glutathione-S-transferase (GSTM1) and microsomal epoxide hydrolase (EH). In a comparison of the two biomarkers, the CA biomarker shows a more definite association with the genotypes than does SCE. For example, the presence of the GSTM1 null genotype (GSTM1 0/0) is responsible for the highest level and significant induction of CA, irrespective of the presence of other genotypes in the different donors. This effect is further enhanced significantly by the presence of the excessive activation EH gene allele (EH4 * ) and decreased by the reduced activation EH gene allele (EH3 * ). Overall, the modulation of genotoxicity by the susceptibility genotypes provides support of their potential involvement in environmental cancer. Furthermore, the data indicate that the variant enzymes function independently by contributing their metabolic capability toward the expression of biologic activities. Therefore, studies like this one can be used to resolve the complexity of genetic susceptibility to environmental disease in human.

Polymorphisms in chemical metabolizing genes are known to influence individual susceptibility to environmental cancer. We investigated the role of GSTM1 and GSTT1 polymorphisms in modifying the genotoxicity of a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) using the sister chromatid exchange (SCE), and the tandem-probe fluorescence in situ hybridization chromosome aberration (CA) assays. NNK (0.24, 0.72 or 1.44 mM) induced a significant concentration-dependent increase in the mean number of SCE regardless of genotypes. In comparing the effects between genotypes, significant increase was observed in GSTM1 null cells compared with GSTM1 positive cells only at the low concentration of NNK (0.24 mM). No significant difference was observed between cells with the null and positive GSTT1 genotypes. Using the CA assay, treatment with NNK (0.12, 0.24 or 0.72 mM) induced a significant concentration-dependent increase in the frequency of CA. In addition, cells with the null GSTM1 genotype had significantly increased CA compared with cells with GSTM1 positive genotype at the three concentrations of NNK. Regarding GSTT1 polymorphism, no significant effect was observed between the null and the positive genotypes. Treatment of the cells with 1 mM glutathione monoethyl ester (GSHME) significantly reduced NNK-induced CA in all cells regardless of their genotypes. The effect was clearly more evident in cells with the GSTM1 positive genotype. Therefore, GSHME is protective against NNK-induced CA with more dominant effect in cells with the GSTM1 positive genotype. Our study indicates that GSTM1 may influence NNK-induced genotoxicity and subsequent tobacco-related health effects.

Inter-individual variation in metabolism of environmental toxicants, which is attributed to genetic polymorphism, may be a major risk factor in determining who will develop adverse health effects. This priority research area is the focus of many laboratories, and new techniques need to be developed to enhance the efficiency in generating data. We have developed and validated a new multiplex-polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) procedure for simultaneous genotyping of cytochrome P450 II E1 (CYP2E1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase mu (GSTM1). Enzymes from these three polymorphic genes are involved with the phase I and II metabolism of a variety of environmental toxicants. Therefore, simultaneous characterization of these genes will not only reduce costs but will increase the efficiency of data collection, thereby contributing to health risk assessment efforts.

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

A fully automated reversed-phase HPLC method was developed for the quantitative assay of three retinoids (Am-580, CD-2019 and CD-437) which selectively activate the retinoic acid receptors RAR alpha, RAR beta and RAR gamma, respectively. Mouse plasma, embryo and maternal tissues were prepared for injection by on-line solid-phase extraction (SPE) and valve-switching techniques. Following automatic injection, the sample was loaded on preconditioned disposable cartridges, cleaned-up and then transferred onto the analytical column to be eluted in the backflush mode, separated by gradient elution and detected by UV, while a new cartridge was concomitantly conditioned. The overall recovery was quantitative allowing for external standardization. The calibration curves were linear in all biological samples tested so far, with a correlation coefficient (r)0.99. The intra-day precision wasor = 7.8% (n = 5-6) and the inter-day variability wasor = 9.4% (n = 3). The lower limit of detection was 2.5 ng/ml or ng/g for CD-2019 and CD-437, and 5 ng/ml for Am-580 with a S/N ratio of 5 using a sample weight of 25 microliters or mg. The method is now in routine use in our laboratory for the assessment of the pharmacokinetic profiles of these retinoids. The small sample size required, the simple sample preparation and the rapid analysis with high degree of automation make this method convenient for microanalysis of biological samples both in animal and human studies.

In the present study, we have addressed the possible protective role of acetyl-L-carnitine in caerulein-induced acute pancreatitis in male Swiss albino rats. Acute pancreatitis paradigm was developed by challenging animals with a supramaximal dose of caerulein (20 microg/kg, SC) four times at hourly intervals. Caerulein induced acute pancreatitis that was well-characterized morphologically and biochemically. Severe oedema with marked increased relative pancreatic weight, marked atrophy of acini with increased interacinar spaces, vacuolization, and extensive leucocytic infiltration were diagnostic fingerprints of the pancreatitis phenotype. A biochemical test battery that confirmed the model comprised increased plasma amylase and lipase activities, calcium levels as well as increased pancreatic enzymatic myeloperoxidase and glutathione-S-transferase activities, beside increased pancreatic contents of nitric oxide and malondialdehyde and reduced pancreatic glutathione level. Prior administration of acetyl-L-carnitine (200 mg/kg, IP) for seven consecutive days ahead of caerulein challenge alleviated all the histological and biochemical manifestations of acute pancreatitis. These results suggest a possible protective role of the carnitine ester in such a murine acute pancreatitis model probably via regulation of the oxidant/antioxidant balance, beside modulation of the myeloperoxidase and nitric oxide systems, which are involved in the inflammatory cascade that most often associate the disease.

Objective To evaluate the utility of gene therapy for uterine fibroids in the Eker rat model using an adenovirus-mediated delivery of a dominant-negative estrogen receptor gene (Ad-DNER). Design Animal study. Setting University animal laboratory. Animal(s) Twenty-seven female Eker rats. Intervention(s) We randomized Eker rats with magnetic resonance imaging (MRI)–confirmed uterine leiomyomas to a single treatment of direct intrafibroid injection with Ad-DNER, Ad–bacterial s-galactosidase, or vehicle. Main Outcome Measure(s) Tumor volumes were determined by MRI scanning and caliper measurement. Samples of serum, fibroid tumors, and various organs were collected at 8, 15, and 30 days after treatment to assess treatment safety and efficacy. Result(s) The Ad-DNER treatment significantly decreased uterine fibroid volume by 45%, 80%, and 77.4% of pretreatment volume at days 8, 15, and 30, respectively, and modulated the expression of apoptosis-, proliferation-, and extracellular matrix–related genes' compared with control animals. The Ad-DNER did not produce any toxic effects in nontarget tissues. Conclusion(s) The Ad-DNER treatment shrinks Eker rats' fibroids, in part, via modulation of several estrogen-regulated genes. This safe gene therapy approach presents a promising conservative treatment option for women with symptomatic uterine fibroids.

Objective This study was undertaken to develop a representative murine model for human leiomyoma. Study Design Human fibroid tumor tissues were cut into small pieces and treated with medium alone, adenoviral-β-galactosidase, adenoviral-vascular endothelial growth factor-A, adenoviral-cyclooxygenase-2, or both adenoviral-vascular endothelial growth factor-A and adenoviral- cyclooxygenase-2. Tissue pieces were inserted subcutaneously in the flank of each severe combined immunodeficient mouse. The developed lesion was measured twice per week. Xenograft tissues were harvested after 30 days and analyzed. Results Tissue pieces transfected with both adenoviral-cyclooxygenase-2 and adenoviral-vascular endothelial growth factor-A continued to grow up to 30 days postimplantation. The number of proliferating and apoptotic cells, as well as the expression of smooth muscle actin, desmin, vimentin, estrogen receptors, and progesterone receptors was similar between retrieved grafts from that group and the original patient tissue. Furthermore, hematoxylin and eosin and Masson's Trichrome stains confirmed this similarity. Conclusion Human uterine leiomyoma xenografts, pretreated with both adenoviral- cyclooxygenase-2 and adenoviral-vascular endothelial growth factor-A and implanted subcutaneously in severe combined immunodeficient mice, represent a novel model for human uterine leiomyoma.

A murine lung fibrosis model has been induced by challenging male Swiss albino mice with a fibrotic dose of bleomycin (10 mg/kg body weight, s.c.) twice weekly for 6 weeks. The model has been characterized and confirmed biochemically, histologically and morphometrically. Keeping in mind that inflammation is the forerunner of lung fibrosis, we have investigated the possible anti-fibrotic effect of meloxicam; a selective COX-2 inhibitor, in this lung fibrosis paradigm. When administered ahead of bleomycin challenge, meloxicam significantly reduced the lung content of hydroxyproline; the backbone of collagen matrix. This was further confirmed by the lower collagen deposition as revealed by histochemical examination of lung sections. Meloxicam had also anti-oxidant effect as shown by increase in lung reduced glutathione (GSH) level and decreases in lung malonedialdehyde (MDA) content and myeloperoxidase (MPO) activity. Besides, meloxicam has shown an apparent angiostatic activity. Histologically, meloxicam lessened lung inflammation and fibrotic changes induced by bleomycin. Taken together, one could conclude that meloxicam has shown anti-fibrotic effect in the bleomycin lung fibrosis model. Apart from its well-known anti-inflammatory potential, this anti-fibrotic action of meloxicam resides most probably, at least partly, in its anti-oxidant and angiostatic effects.

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

The effects of benzo[a]pyrene (B[a]P) on some drug-metabolizing and antioxidant systems in liver, lung, and stomach were investigated in normal and protein malnutrition (PM) rats. PM significantly inhibited tissue glutathione (GSH) content and increased hepatic lipid peroxidation. Cytochrome P450 isoform CYP1A1 was significantly increased in various tissues (42-73%). Also, lung glutathione S-transferase (GST) activity was significantly decreased (19%) in PM rats. On the other hand, B[a]P significantly induced tissue GSH of control and PM rats. Also, hepatic lipid peroxidation were significantly increased in control rats treated with B[a]P. Superoxide dismutase (SOD) activity was decreased by B[a]P treatment in PM rat stomach. B[a]P significantly induced both quinone reductase (QR) (in all tissues) and hepatic GST of control and PM rats. GST activity in PM rat liver was significantly higher than that of control rat liver after B[a]P treatment. Also, B[a]P induced hepatic CYP1A1 by 32-fold and 27-fold (P < or = 0.05) in control and PM rats, respectively. Stomach and hepatic UDP-glucuronosyltransferase activities were significantly decreased (34%) and increased (74%), respectively by B[a]P in PM rats. The results suggest that PM status has a modifying effect on the response of some antioxidant and metabolizing systems to a well-known carcinogen risk.

Atherosclerosis (AR) is the leading cause of morbidity and mortality in the US and cigarette smoking is a major contributing factor to the disease. Like cigarette smoking in lung cancer, genetic susceptibility may be an important factor in determining who is more likely to develop AR. However, the current emphasis has been on susceptibility based on altered cardiovascular homeostasis. In this investigation, we studied 120 AR patients and 90 matched controls to elucidate the association between polymorphisms in some metabolizing genes (GSTM1, GSTT1, CYP2E1, mEH, PON1, and MPO) and susceptibility to AR. We found that the GSTT1 null allele and the fast allele of mEH(*) (exon 4) are associated with risk for AR. Furthermore, the combined genotypes GSTM1 null/ CYP2E1(*)5B, GSTM1 null/mEH YY, and GSTT1 null/mEH YY are significantly associated with susceptibility to AR (OR = 15.42, 95% CI = 1.33-77.93, P = 0.021; OR = 3.48, 95% CI = 1.63-8.04, P = 0.0008; OR = 3.4; 95% CI = 0.99-17.38, P = 0.05; respectively). We have also conducted cytogenetic analysis to elucidate if induction of chromosome aberrations (CAs) is a biomarker of AR susceptibility. We found that among cigarette smokers (AR patients and smoker controls), individuals having the GSTM1 null allele had a significantly higher frequency of CAs compared to those with the normal allele (P < 0.05). This association was not found among nonsmokers. In addition, individuals who had inherited the CYP2E1(*)5B allele exhibited a significantly higher CA frequency (8.0 +/- 0.82) compared to those with the CYP2E1 wild-type genotype (4.31 +/- 0.35). Since the analysis of genetic susceptibility factors is still in its infancy, our study may stimulate additional investigations to understand the roles of genetic susceptibility and cigarette smoking in AR.

The effect of thymoquinone (TQ), the main constituent of the volatile oil of Nigella sativa seeds, on the nephropathy and oxidative stress induced by doxorubicin (DOX) in rats was investigated. A single intravenous injection of DOX (6 mg/kg) induced a severe nephrotic syndrome (after 5 weeks) associated with hypoalbuminemia, hypoproteinemia, elevated serum urea, hyperlipidemia, and a high urinary excretion of protein, albumin and N -acetyl-β- d -glucosaminidase (NAG). In the kidney, DOX induced a significant increase in total triglycerides (TG), total cholesterol (TC), and lipid peroxides and a significant decrease in non-protein sulfhydryl (NPSH) content and catalase (CAT) activity. Treatment of rats with TQ (10 mg/kg per day) supplemented with the drinking water for 5 days before DOX, and daily thereafter, significantly lowered serum urea, TG, and TC. Similarly, TG, TC and lipid peroxides in the kidneys of TQ-treated rats were decreased significantly compared with DOX alone. Moreover, NPSH content and CAT activity in the kidneys of TQ-treated DOX group were significantly elevated compared with DOX alone. Treatment with TQ significantly suppressed DOX-induced proteinuria, albuminuria, and urinary excretion of NAG. The results confirm the involvement of free radicals in the pathogenesis of nephropathy induced by DOX. Likewise, the study demonstrates the high antioxidant potential of TQ and its marked effect on the suppression of DOX-induced nephropathy. The data suggest that TQ might be applicable as a protective agent for proteinuria and hyperlipidemia associated with nephrotic syndrome.

Ofloxacin induces its antibacterial action mainly by inhibition of DNA gyrase which is equivalent to topoisomerase II in mammalian cells. The present study was focused on the impact of ofloxacin on rat testicular DNA ploidy in a dose–response relationship using an image analysis technique on testicular sections following Fuelgen DNA staining. Sperm count and motility as well as sperm head abnormality tests were also investigated. Ofloxacin was given p.o. in doses of 36, 72 and 360 mgkg −1 day −1 for 15 consecutive days. The animals were then left to live safely to day 60 from starting the experiment to give them a chance to complete the cycle of spermatogenesis. Results revealed that ofloxacin significantly decreased the percentage of haploid cells in a dose-dependent manner with concomitant increase in the percentage of diploid cells. Sperm count and motility was also markedly decreased in a dose-dependent fashion. Sperm head abnormality showed no marked change following ofloxacin treatment. In conclusion, ofloxacin induced a marked disturbance in rat testicular DNA ploidy, which may be explained on the basis of cross-reactivity to topoisomerase II.

Aims and background Nausea and vomiting occur in the majority of patients receiving cisplatin (CDDP) chemotherapy. Ondansetron, a new 5-HT3 receptor antagonist, has been used effectively to control CDDP-induced nausea and vomiting. This study examined the potential of ondansetron to interfere with CDDP antitumor activity and toxicity in Ehrlich ascites carcinoma (EAC). Methods The influence of ondansetron on CDDP cytotoxicity was evaluated using EAC cells in culture. In addition, the influence of ondansetron pretreatment on CDDP-induced antitumor activity and host tissue toxicity was studied in EAC-bearing mice. Results Ondansetron (0.25 μM) enhanced CDDP (0–32 μM) cytotoxicity against EAC cells in vitro. In EAC-bearing mice ondansetron (0.2 mg/kg, ip) administered 1 h before CDDP (7 mg/kg, ip) did not modify the antitumor activity of CDDP. CDDP (7 mg/kg, ip) single treatment induced significant increases in blood urea nitrogen (2-fold) and serum creatinine (2.5-fold) and significant decreases in hematocrit (25%) and white blood cell count (39%) compared to saline treatment. Mice receiving ondansetron 1 h before CDDP showed no significant enhancement of CDDP-induced nephrotoxicity or myelosuppression compared to those pretreated with saline receiving the same dose of CDDP. Conclusions This study suggests that the use of ondansetron to control CDDP-induced nausea and vomiting does not affect CDDP antitumor efficacy.

Polymorphisms in genes of xenobiotic-metabolizing enzymes are largely responsible for interindividual differences in ability to activate and detoxify genotoxic agents and therefore may influence individual susceptibility to environmental cancer. The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), requires metabolic activation by cytochrome P450 (CYP) enzymes to generate DNA-reactive intermediates that induce mutations and cancer. In the current study, we investigated the role of the polymorphic CYP2E1 and CYP2D6 genes in the genotoxicity of NNK using the tandem-probe fluorescence in-situ hybridization (FISH) chromosome aberration assay as a marker. Our results, using whole blood cultures from 39 volunteers, indicated that NNK (0.12, 0.24 or 0.72 mM) induced a concentration-dependent increase in the frequency of chromosome aberration. The potential role of CYP2E1 and CYP2D6 in NNK-induced genetic damage in cultured human lymphocytes was characterized using specific CYP inhibitors. Treatment of blood cultures with 25 microM diethyldithiocarbamate (DDC), a specific CYP2E1 inhibitor, or 0.5 microM quinidine, a specific CYP2D6 inhibitor, simultaneously with NNK, significantly decreased NNK-induced chromosome aberration. We also studied the role of CYP2E1 and CYP2D6 allelic variants on NNK-induced chromosome aberration. Our results indicate that NNK induced a significantly higher level of chromosome aberration in cells with the CYP2E1 WT/*5B genotype compared to cells with the CYP2E1 WT/WT. In contrast, no difference in NNK-induced chromosome aberration was observed between cells with the CYP2D6 extensive metabolizers compared to cells with the CYP2D6 poor metabolizer genotypes. These results underscore the important role of polymorphic metabolizing genes in influencing the genotoxic responses to environmental mutagens and provide support to the reported findings linking CYP2E1 polymorphism to smoking-related lung cancer.

Cremophor EL (CR), the paclitaxel vehicle, has previously been reported to alter the pharmacokinetics and/or pharmacodynamics of some anticancer drugs including paclitaxel. Several experimental and clinical studies suggested that cisplatin (CDDP) in combination with paclitaxel results in less hematological toxicity than anticipated. To reveal the role of CR in this important pharmacological interaction, we evaluated the interaction of CR with CDDP in vitro and in vivo using experimental Ehrlich ascites carcinoma (EAC) tumor. CR (1 microg/ml) significantly enhanced the in vitro cytotoxicity of CDDP in cultured EAC cells. This enhancement was not associated with a parallel increase in CDDP cellular uptake. In tumor-bearing mice, CR (2.5 ml/kg, i.v.) given in combination with CDDP (7 mg/kg, i.v.) did not significantly change CDDP pharmacokinetics, antitumor activity or nephrotoxicity. On the other hand, CDDP-induced hematological toxicity was significantly reduced by CR. This protective effect was related to CR-induced inhibition of cellular CDDP accumulation in bone marrow. This study presents evidence that CR may play an important role in the pharmacological interaction between CDDP and paclitaxel. The present data may suggest formulation of CDDP with CR for systemic treatment. Further studies are yet necessary to establish the clinical value of CR as a modifier for CDDP therapeutic index.

Three biologically active synthetic retinoids were investigated that bind selectively to retinoic acid receptors RARs (alpha, beta and gamma). The retinoids were previously demonstrated to have different teratogenic effects in the mouse in terms of potency and regioselectivity. The teratogenic potency rank order (alphabetagamma) was found to be more or less compatible with the receptor binding affinities and transactivation potencies of the retinoid ligands to their respective receptors. The RARalpha agonist (Am580; CD336) induced a wide spectrum of malformations; CD2019 (RARbeta agonist) and especially CD437 (RARgamma agonist) produced more restricted defects. In the current study we tried to address whether the differences in teratogenic effects are solely related to binding affinity and transactivation differences or also due to differences in embryonic exposure. Therefore, transplacental kinetics of the ligands were assessed following administration of a single oral dose of 15 mg/kg of either retinoid given to NMRI mice on day 11 of gestation. Am580 was rapidly transferred to the embryo resulting in the highest embryonic exposure [embryo to maternal plasma area under the time vs concentration curve (AUC)(0-24 h )ratio (E/M) was 1.7], in accordance with its highest teratogenic potency. The low placental transfer of CD2019 (E/M of 0.3) was compatible with its lower teratogenic potential. Of major interest was the finding that the CD437, though being least teratogenic, exhibited considerable embryonic exposure (E/M of 0.6). These findings suggest that both the embryonic exposure and receptor binding transactivation selectivity are crucial determinants of the teratogenicity of these retinoid ligands.

To study the effects of ferulic acid on gastrointestinal motility both in vitro and in vivo.Ferulic acid induced concentration-dependent stimulation of the basal tone of isolated guinea pig ileum (2-20 micromol/L) and isolated rat fundus (0.05-0.4 mmol/L).Ferulic acid significantly accelerated the gastrointestinal transit and gastric emptying in rats in a dose-dependent manner (50-200 mg/kg, po). Cisplatin (2.5-20 mg/kg, ip) induced a dose-dependent delay in gastric emptying in rats. Pretreatment with ferulic acid dose-dependently, significantly reversed the cisplatin-induced delay in gastric emptying.The endogenous prostaglandins (PGs) are involved in mediating the stimulant effects of ferulic acid. This effect of dietary ferulic acid may help improve other accompanying gastrointestinal symptoms such as abdominal discomfort and also may protect against emesis induced by cytotoxic drugs.