Memy H. Hassan

Crocin “saffron” protects against beryllium chloride toxicity in rats through diminution of oxidative stress and enhancing gene expression of antioxidant enzymes

Beryllium chloride (BeCl(2)) is a highly toxic substance that accumulates in different tissues after absorption. The purpose of this study was to investigate protective role of crocin against BeCl(2)-intoxication in rats. Male Wistar rats were used in this study and categorised into four groups (n=8). Group I served as normal control rats. Group II treated orally with BeCl(2) 86 mg/kg b.w. for five consecutive days. This dose was equivalent to experimental LD(50). Group III treated intraperitoneally with crocin 200 mg/kg b.w. for seven consecutive days. Group IV received crocin for seven consecutive days before BeCl(2) administration. Blood samples and liver and brain homogenates were obtained for haematological, biochemical and RT-PCR examinations. The haematocrit value, RBCs count and haemoglobin concentration were significantly decreased in BeCl(2)-treated rats. A significant increase was observed in rat liver and brain malondialdehyde level and protein carbonyls content in BeCl(2) exposed group compared to the control group, and these values were significantly declined upon administration of crocin. Lactate dehydrogenase levels in rat liver and brain significantly increased compared to the control group and was associated with significant decrease in catalase and superoxide dismutase activities. Reduced glutathione hepatic contents of BeCl(2)-treated rats were significantly decreased. There was significant decline in mRNA expression of catalase and superoxide dismutase genes in BeCl(2)-intoxicated rats compared to the normal rats. Crocin treatment prior to BeCl(2) intake resulted in significant increase in mRNA expressions of catalase and superoxide dismutase genes near to normalcy. The haematological and biochemical parameters were restored near to normal levels. Our results suggested that, BeCl(2) induced oxidation of cellular lipids and proteins and that administration of crocin reduced BeCl(2)-induced oxidative stress combined with initiation of mRNA expression of antioxidant genes.

Dibutyl phthalate induces oxidative stress and impairs spermatogenesis in adult rats

Phthalates are abundantly produced plasticizers, and dibutyl phthalate (DBP) is the most widely used derivative in various consumer products and medical devices. This study was conducted to further explore the potential testicular toxicity of DBP in adult rats and to elucidate the underlying mechanisms. Adult male albino rats were treated orally with DBP at doses of 0, 200, 400, or 600 mg/kg/day for 15 consecutive days. Testicular weight, sperm count, and motility were significantly decreased. Treatment with DBP decreased serum follicle-stimulating hormone and testosterone levels and testicular lactate dehydrogenase activity. DBP treatment also decreased serum total antioxidant capacity and the activities of the testicular antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase. Further, DBP treatment provoked degeneration with absence of spermatogenesis and sperms and necrosis in some of seminiferous tubules. These results indicated that oxidative stress and subsequent decrease in testosterone secretion were the potential underlying mechanism of DBP-induced testicular toxicity.

Antioxidant and antiapoptotic effects of capsaicin against carbon tetrachloride-induced hepatotoxicity in rats

The aim of the study was to evaluate the potential hepatoprotective utility of capsaicin against carbon tetrachloride (CCl4)-induced liver injury and to explore the possible mechanisms whereby this agent mediated its beneficial effects. We randomized 40 rats into four groups for treatment with corn oil, CCl4, capsaicin and both CCl4 and capsaicin, respectively, for 8 weeks. At the end of the experiment, blood samples were collected and used for determination of aspartylaminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin, while the liver tissues were subjected to hematoxylin and eosin examination; evaluation of malondialdehyde (MDA), reduced glutathione (GSH) and active caspase-3 contents; and evaluation of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) activities. Animals treated with CCl4 exhibited significant elevation in AST, ALT, total bilirubin and caspase-3 and exhibited significant decrease in activities of SOD, CAT, GST and GSH contents. The combination (both capsaicin and CCl4) group has preserved the liver histology, liver enzymes and bilirubin close to normal, exhibited significant induction in the activities of CAT, SOD and GST, increased the liver content of GSH and active caspase-3 and conversely showed significant decrease in liver MDA content compared to CCl4 challenged rats. Capsaicin confers an appealing hepatoprotective effect which might be explained partially via diminishing the generation of MDA, induction of antioxidant systems and inhibition of active caspase-3.

Ameliorative Effects of Resveratrol on Liver Injury in Streptozotocin-Induced Diabetic Rats

The objective of this study was to investigate the ameliorative property and potential mechanism of resveratrol (RVT) in a dose of 10 mg/kg for 15 consecutive days against liver injury in streptozotocin‐induced diabetic rats. Diabetic rats significantly (P < 0.05) exhibited liver injury manifested by increased aspartylaminotransferase, alanine aminotransferase, and bilirubin; disturbed liver weight to body weight; and confirmed by hematoxylin and eosin staining. Liver from diabetic rats exhibited significant increase in malondialdehyde level and significant decrease in reduced glutathione, glutathione‐S‐transferase, quinone reductase, catalase, and superoxide dismutase. Diabetic rats showed significant disturbance in serum lipid profile. Treatment with RVT significantly (P < 0.05) abrogated diabetes‐induced perturbation in these parameters and liver histology. These data suggest that RVT treatment is associated with promising hepatoprotective effect against diabetes‐induced liver damage via reduction of serum glucose level and oxidative damage and improving serum lipid profile.

Memy I: a novel murine model for uterine leiomyoma using adenovirus-enhanced human fibroid explants in severe combined immune deficiency mice

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-beta-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 Massons 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.

Towards Fibroid Gene Therapy: Adenovirus-Mediated Delivery of Herpes Simplex Virus 1 Thymidine Kinase Gene/Ganciclovir Shrinks Uterine Leiomyoma in the Eker Rat Model

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.

Gene therapy of benign gynecological diseases

Gene therapy is the introduction of genetic material into patients cells to achieve therapeutic benefit. Advances in molecular biology techniques and better understanding of disease pathogenesis have validated the use of a variety of genes as potential molecular targets for gene therapy based approaches. Gene therapy strategies include: mutation compensation of dysregulated genes; replacement of defective tumor-suppressor genes; inactivation of oncogenes; introduction of suicide genes; immunogenic therapy and antiangiogenesis based approaches. Preclinical studies of gene therapy for various gynecological disorders have not only shown to be feasible, but also showed promising results in diseases such as uterine leiomyomas and endometriosis. In recent years, significant improvement in gene transfer technology has led to the development of targetable vectors, which have fewer side-effects without compromising their efficacy. This review provides an update on developing gene therapy approaches to treat common gynecological diseases such as uterine leiomyoma and endometriosis.

Towards non-surgical therapy for uterine fibroids: catechol-O-methyl transferase inhibitor shrinks uterine fibroid lesions in the Eker rat model

BACKGROUND Uterine leiomyomas (fibroids) are the most common pelvic tumors in women. We assessed the potential therapeutic utility of Ro 41-0960, a synthetic catechol-O-methyl transferase inhibitor (COMTI), in the Eker rat. METHODS We randomized uterine fibroid-bearing Eker rats for treatment with Ro 41-0960 (150 mg/kg/12 h) versus vehicle for 2 and 4 weeks. The fibroids were measured by caliper and subjected to histological evaluation. Urinary levels of 2-hydroxy estrogen (E(2)), 16-hydroxy E2 and DPD (osteoporosis marker) and serum liver enzymes were evaluated. Expressions of Cyclin D1, proliferating cell nuclear antigen (PCNA), Poly [ADP-ribose] polymerase1 (PARP1), tumor suppressor gene (P53) and transforming growth factor (TGFβ3) were assessed in fibroids using immunohistochemical analysis or RT-PCR. Apoptosis was confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL). RESULTS Ro 41-0960-treated rats exhibited fibroid volumes of 86 ± 7% and 105 ± 12% of initial burden, at 2 and 4 weeks post-treatment, respectively, significantly lower than control group (240 ± 15% and 300 ± 18%; P< 0.01). Ro 41-0960 increased the urinary 2-hydroxy E2/16-hydroxy E(2) ratio, level of p53 mRNA and TUNEL positivity (P< 0.05) and decreased PARP1, PCNA and cyclin D1 proteins and TGFβ3 mRNA (P< 0.05). Ro 41-0960 did not change normal tissue histology, liver functions or urinary DPD level. CONCLUSIONS Ro 41-0960 (COMTI) arrested growth/shrunk uterine fibroids in Eker rats. This result may be related to modulation of estrogen-dependent genes involved in apoptosis, proliferation and extracellular matrix deposition via accumulation of 2-hydroxy estrogen. The efficacy and safety of Ro 41-0960 in rats suggest its candidacy for treatment of uterine fibroids.

Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells

BACKGROUND To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial. METHODS We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content. RESULTS Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels. CONCLUSIONS Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.

Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes

Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.