Antiarthritic activity of OA-DHZ; a gastroprotective NF-κB/MAPK/COX inhibitor.

Arthritis, a primary autoimmune disorder having a global incidence of 2.03% person/year, is presently being treated by many commercially available drugs that treat symptomatically or improve the disease's clinical state; however, all the therapies pose varying amount of side effects. Therefore, it has become a fundamental need to search for therapeutics that offer better efficacy and safety profile, and the natural or nature-derived products are known for their outstanding performance in this arena. OA-DHZ, known to possess anti-inflammatory and analgesic properties, when explored for its efficacy against arthritis in adjuvant-induced arthritis (AIA) model, was found to inhibit paw edema by 34% and TNF-α, IL-6, and IL-1ß by 67%, 39%, and 45% respectively when compared to diseased control. It was also able to reduce the inflamed spleen size by 45% and successfully normalized biochemical and hematological changes that followed arthritis. In vitro studies revealed that the underlying mechanism for inhibiting arthritis progression might be due to NF-κB /MAPK pathway modulation. OA-DHZ also showed selective inhibition of COX-2 in vitro while showing gastroprotective effects when evaluated for ulcerogenic and antiulcer potential in vivo. In contrast to the results obtained from in vivo experimentation, there is a disparity in the pharmacokinetic profile of OA-DHZ, where it showed low oral exposure and high clearance rate. OA-DHZ being antiarthritic acting via NF-κB /MAPK/ COX inhibition while showing gastroprotective effects, can be a suitable candidate to be in the drug pipeline and further exploration.

Drug-in-hydroxypropyl-ß-cyclodextrin-in-lipoid S100/cholesterol liposomes: Effect of the characteristics of essential oil components on their encapsulation and release.

Drug-in-cyclodextrin-in-liposome (DCL) represents a very promising approach for preserving essential oil (EO) components, thereby extending their shelf life and activity. In this study, we examined the effect of chemical structure, octanol/water partition coefficient (log P), and Henry's law constant (Hc) on the encapsulation and the release of monoterpenes (eucalyptol, pulegone, terpineol, and thymol) and phenylpropenes (estragole and isoeugenol) from DCLs. Hydroxypropyl-ß-cyclodextrin/EO component (HP-ß-CD/EO component) inclusion complexes were prepared in aqueous solution and loaded into liposomes by the ethanol injection method. The phospholipid:cholesterol:EO component molar ratio determined for DCL structures was affected by characteristics of EO components. The presence of a propenyl tail or a hydroxyl group in the structure of EO component may improve its loading into DCLs. Furthermore, low encapsulation efficiency (EE) was obtained for DCLs exhibiting high cholesterol membrane content. In addition, a positive linear relationship was found between the loading ratio of monoterpenes into DCLs and their hydrophobic character expressed as log P. The release of components from DCLs was influenced by their EE into the formulations. Finally, DCL formulations retain considerable amounts of EO components after 10 months.

Anti-Inflammatory Potential of 1-Nitro-2-Phenylethylene.

Inflammation is a reaction of the host to infectious or sterile stimuli and has the physiological purpose of restoring tissue homeostasis. However, uncontrolled or unresolved inflammation can lead to tissue damage, giving rise to a plethora of chronic inflammatory diseases, including metabolic syndrome and autoimmunity pathologies with eventual loss of organ function. Beta-nitrostyrene and its derivatives are known to have several biological activities, including anti-edema, vasorelaxant, antiplatelet, anti-inflammatory, and anticancer. However, few studies have been carried out regarding the anti-inflammatory effects of this class of compounds. Thereby, the aim of this study was to evaluate the anti-inflammatory activity of 1-nitro-2-phenylethene (NPe) using in vitro and in vivo assays. Firstly, the potential anti-inflammatory activity of NPe was evaluated by measuring TNF-α produced by human macrophages stimulated with lipopolysaccharide (LPS). NPe at non-toxic doses opposed the inflammatory effects induced by LPS stimulation, namely production of the inflammatory cytokine TNF-α and activation of NF-κB and ERK pathways (evaluated by phosphorylation of inhibitor of kappa B-alpha [IκB-α] and extracellular signal-regulated kinase 1/2 [ERK1/2], respectively). In a well-established model of acute pleurisy, pretreatment of LPS-challenged mice with NPe reduced neutrophil accumulation in the pleural cavity. This anti-inflammatory effect was associated with reduced activation of NF-κB and ERK1/2 pathways in NPe treated mice as compared to untreated animals. Notably, NPe was as effective as dexamethasone in both, reducing neutrophil accumulation and inhibiting ERK1/2 and IκB-α phosphorylation. Taken together, the results suggest a potential anti-inflammatory activity for NPe via inhibition of ERK1/2 and NF-κB pathways on leukocytes.

Synthesis and evaluation of styrylpyran fluorophores for noninvasive detection of cerebral ß-amyloid deposits.

The development of amyloid-specific fluorophores allows the visualization of cerebral ß-amyloid deposits using optical imaging technology. In the present study, a series of smart styrylpyran fluorophores with compact donor-acceptor architecture were designed and evaluated for noninvasive detection of cerebral ß-amyloid deposits. Spectral behavior of the fluorophores changed significantly (optical turn-on) upon binding to ß-amyloid aggregates. Computational studies were conducted to correlate the experimental Kd values with calculated binding energies, speculating the relationship between fluorophore structure and ß-amyloid affinity. In vivo studies demonstrated that PAD-2 could discriminate APP/PS1 transgenic mice from wild type controls, with specific labeling of cerebral ß-amyloid deposits confirmed by ex vivo observation. Collectively, these styrylpyran fluorophores could provide a new scaffold for the development of optical imaging probes targeting cerebral ß-amyloid deposits.

Dual inhibition of EGFR and c-Met kinase activation by MJ-56 reduces metastasis of HT29 human colorectal cancer cells.

Quinazolinone derivatives are known to possess anticancer activities on cell metastasis and cell death in different human cancer cell lines. Here, we studied the anti-metastasis activity and the underlying mechanisms of the novel quinazoline derivative MJ-56 (6-pyrrolidinyl-2-(3-bromostyryl)quinazolin-4-one). MJ-56 inhibited cell migration and invasion of HT29 human colorectal cancer cells by wound-healing and Matrigel-coated transwell assays in a concentration-dependent manner. MJ-56-treated cells resulted in the reduced expression of matrix metalloproteinase (MMP)-2, -7, -9 and -10 and the reduced enzymatic activities of MMP-2 and MMP-9. In contrast, MJ-56-treated cells enhanced the expression of the tissue inhibitors of metalloproteinases (TIMPs) TIMP-1 and TIMP-2. Further analyses showed that MJ-56 attenuated the activities of epidermal growth factor receptor (EGFR), c-Met and the downstream ERK-mediated MAPK and PI3K/AKT/mTOR signaling pathways, which led to decreased protein synthesis by dephosphorylating the translation initiation factors eIF-4B, eIF-4E, eIF-4G and S6 ribosomal protein. In addition, MJ-56 interfered with the NF-κB signaling via impairing PI3K/AKT activation and subsequently reduced the NF-κB-mediated transcription of MMPs. Taken together, the reduced expression of phosphor-EGFR and c-MET is chiefly responsible for all events of blocking metastasis. Our results suggest a potential role of MJ-56 on therapy of colorectal cancer metastasis.

Combined administration of rituximab and on 013105 induces apoptosis in mantle cell lymphoma cells and reduces tumor burden in a mouse model of mantle cell lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma, and new therapeutic strategies are urgently needed. EXPERIMENTAL DESIGN: The effects of ON 013105, a novel benzylstyryl sulfone kinase inhibitor, alone or with doxorubicin or rituximab, were examined in Granta 519 and Z138C cells. For in vivo studies, CB17/SCID mice were implanted subcutaneously with Z138C cells and treated with various combinations of ON 013105, doxorubicin, and rituximab. Tumor burden and body weight were monitored for 28 days. RESULTS: ON 013105 induced mitochondria-mediated apoptosis in MCL cells. Death was preceded by translocation of tBid to the mitochondria and cytochrome c release. In addition, ON 013105-treated cells exhibited reduced levels of cyclin D1, c-Myc, Mcl-1, and Bcl-xL. Using nuclear magnetic resonance (NMR) spectroscopy, we showed specific binding of ON 013105 to eIF4E, a critical factor for the initiation of protein translation. We proffer that this drug-protein interaction preferentially prevents the translation of the aforementioned proteins and may be the mechanism by which ON 013105 induces apoptosis in MCL cells. Efficacy studies in a mouse xenograft model showed that ON 013105 inhibited MCL tumor growth and that combining ON 013105 with rituximab reduced tumor burden further with negligible unwanted effects. CONCLUSIONS: Our findings suggest that ON 013105, alone or in combination with rituximab, may be a potent therapeutic agent to treat MCLs.

Polyplexes based on cationic polymers with strong nucleic acid binding properties.

Cationic polymers have been studied for nucleic acid delivery both in vitro and in vivo. However, many polymer-based formulations suffer from lack of stability in biologic fluids due to interactions with anionic biomacromolecules such as proteins and polysaccharides. Likely, the stronger the electrostatic interactions between a cationic polymer and nucleic acids, the higher the stability of the polyplexes in biologic fluids will be. To get evidence for this hypothesis, quaternized poly[3,5-bis(dimethylaminomethylene)-p-hydroxyl styrene] (QNPHOS) with two permanently charged cationic sites per monomer unit as well as its block copolymer with PEG were synthesized and compared with the standard transfectant pDMAEMA, in terms of nucleic acid binding strength, gene silencing and transfection activities of the complexes which these polymers form with siRNA and plasmid DNA, respectively. It was shown that siRNA complexes based on QNPHOS and QNPHOS-PEG dissociate in the presence of a fourfold higher heparin concentration than necessary to destabilize pDMAEMA complexes. Under the same conditions, complexes of DNA and QNPHOS or QNPHOS-PEG did not show any dissociation, in contrast to pDMAEMA polyplexes. The DNA polyplexes based on QNPHOS or QNPHOS-PEG did not show transfection activity, which might be ascribed to their high physicochemical stability. On the other hand, siRNA complexes based on QNPHOS and QNPHOS-PEG showed a low cytotoxicity and an improved siRNA delivery and high gene silencing activity, even higher than those based on pDMAEMA. This might be due to the excellent binding characteristics of QNPHOS and QNPHOS-PEG to siRNA which in turn is ascribed to the presence of two permanently charged cationic groups per monomer unit. Based on the results of this study, it is concluded that formation of strong siRNA complexes with polymers containing double charges per monomer is advantageous.

1,3-butadiene, styrene and lung cancer among synthetic rubber industry workers.

OBJECTIVE: To evaluate the association between cumulative exposure to 1,3-butadiene (BD) or styrene (STY) and lung cancer among synthetic rubber industry workers. METHODS: Internal Cox regression analyses were performed both with and without natural logarithm transformation of exposure variables and both with and without the inclusion of those unexposed to monomers. RESULTS: Among women, analyses using untransformed BD exposure showed no trend. Analyses using natural logarithm-transformed BD exposure indicated a positive trend when the unexposed were included (P < 0.05) and an inverse trend when the unexposed were excluded (P < 0.05). No exposure-response trends were seen for BD among men or for STY among women or men. CONCLUSIONS: These results suggest that the association between BD and lung cancer, seen in some analyses of female employees, is not causal. STY did not appear to be associated with lung cancer.

A follow-up study of mortality among women in the North American synthetic rubber industry.

OBJECTIVE: To evaluate mortality from cancer and other diseases among 4863 women employed at eight North American styrene-butadiene rubber plants. Cancers of the lymphohematopoietic tissues, breast, and ovary were of strongest a priori interest. METHODS: Cause-specific standardized mortality ratios (SMRs) and 95% confidence intervals (CIs) were estimated. RESULTS: The observed number of deaths was approximately equal to that expected for leukemia (10 observed/13 expected), Hodgkin lymphoma (1/1.6), multiple myeloma (7/7.9), non-Hodgkin lymphoma (15/14), and cancers of the breast (72/74) and ovary (21/22). Ever-hourly women had more than expected deaths from lung (47/30, SMR = 159, CI = 117 to 211) and bladder (6/1.8, SMR = 332, CI = 122 to 723) cancers. Exposure-response analysis, done only for lung cancer, indicated no trend for butadiene or styrene. CONCLUSIONS: The observed excesses of lung and bladder cancers may be attributable to nonoccupational factors rather than to workplace exposures.

Epidemiologic studies of styrene and cancer: a review of the literature.

OBJECTIVE: To review the epidemiologic literature on styrene and cancer. METHODS: We reviewed studies of workers exposed to styrene in manufacturing and polymerization, in the reinforced plastics industry, and in styrene-butadiene rubber production. We also reviewed studies of workers monitored for styrene exposure, studies of environmental exposure, community-based case-control studies of lymphoma and leukemia, and studies of DNA adducts. Studies of workers in the reinforced plastics industry were considered more informative because of higher worker exposure and less confounding by other carcinogens. RESULTS: We found no consistent increased risk of any cancer among workers exposed to styrene. A study of reinforced plastic workers reported an association between average estimated styrene exposure and non-Hodgkin lymphoma (NHL, P = 0.05) but no trend with increasing duration of exposure. Other studies of styrene exposure and NHL found no increased risk. In two US studies of reinforced plastic workers, esophageal cancer mortality was increased, but these findings were generated in a background of multiple comparisons. Results for other cancers were unremarkable. CONCLUSIONS: The available epidemiologic evidence does not support a causal relationship between styrene exposure and any type of human cancer.

Whole genomic expression analysis of octachlorostyrene-induced chronic toxicity in Caenorhabditis elegans.

In recent years, microarray technology has enabled the investigation of possible mechanisms the expression of genes related to toxic compounds. We used a C. elegans whole genome microarray to observe and evaluate the chronic toxicity of the free-living nematode Caenorhabditis elegans (C. elegans) after exposure to octachlorostyrene, (OCS), a by-product in the manufacture of many chlorinated hydrocarbons. In this study, we examined sublethal toxicity, egg hatching, and movement of octachlorostyrene over three generations using a nematode growth medium (NGM) agar plate. In the third generation, OCS affected the fecundity rate of C. elegans. Specifically, the number of worm and eggs decreased significantly to about 50% of control (p < 0.05). In microarray experiments, total RNA was isolated at 0, 2 and 3 generations following treatment of OCS, and hybridized to the microarray containing about 22,000 C. elegans genes. Dye swaps were performed. After data analysis, we identified a total of 1,294 genes that were differentially expressed through generations.

Hypotensive and antiaggregative effects of eugenosedin-B with serotonin and alpha/beta-adrenoceptor antagonistic activities in rats and human platelets.

Eugenosedin-B is able to block serotonin (5-HT) and alpha/beta receptors and to inhibit platelet aggregation. In Wistar rats, intravenous injections of eugenosedin-B (2.4, 7.2, 12 micromoL/kg) caused a dose-dependent decrease in blood pressure and heart rate. In contrast, intracisternal injection of eugenosedin-B (0.3, 0.03 micromoL) and an alpha2-antagonist yohimbine (0.03 micromoL) increased blood pressure and heart rate. Eugenosedin-B and yohimbine prevented hypotension induced by intracisternal injection of an alpha2-agonist clonidine (38 pmol). In in vitro experiments, eugenosedin-B (10, 10, 10 M) competitively antagonized norepinephrine-, clonidine-, and 5-HT (10 to 10 M)-induced vasocontractions in isolated rat aorta. It also competitively antagonized the isoproterenol (10 to 10 M)-induced positive inotropic effects in isolated rat atrium. These findings clearly suggest that eugenosedin-B possesses alpha1, alpha2, beta1, and 5-HT2A receptor blocking activities. In isolated rabbit ear artery sensitized with 16 mM K, eugenosedin-B antagonized 5-nonyloxytryptamine- and 5-HT-induced vasocontractions, indicating it also blocked 5-HT1B and 5-HT2A receptors. In radioligand-binding experiments, eugenosedin-B had significant binding affinities on alpha1, alpha2, beta1, 5-HT1B, and 5-HT2A receptors. In human platelets, eugenosedin-B inhibited epinephrine and 5-HT-induced aggregations. It also had competitive binding effects in human platelet with [H]yohimbine (alpha2), [H]ketanserin (5-HT2A). We conclude that hypotensive and vasorelaxant effects of eugenosedin-B can be attributed to its multiple actions on the blockade of 5-HT1B, 5-HT2A, alpha1/2 and beta1 receptors, and its ability to reduce platelet aggregation attributed to its blockade of alpha2 and 5-HT2A receptors.

A microscopic technique to study kinetics and concentration-response of drug-induced caspase-3 activation on a single cell level.

INTRODUCTION: Induction of apoptosis is perceived as the main intention of drug regimens for tumour therapy. Thus, the concentration- and time-dependence of drug-induced apoptosis should be carefully evaluated for experimental as well as for standard anti-tumour agents. A main feature of apoptosis is the activation of caspases which is a specific phenomenon of the individual cell. Since caspase-3 is one of the key enzymes we developed a fluorescence microscopy technique to detect caspase-3 activity on the single cell level. The results obtained with this technique were compared to a biochemical procedure investigating caspase-3 activation in a cell population. METHODS: For the single cell assay LoVo adenocarcinoma cells were stably transfected with the vector pCaspase3-Sensor. The activated caspase-3 cleaves the cytosolic fusion protein and its EYFP part translocates into the nucleus. Thus, each individual apoptotic cell displays a labelled nucleus and affected cells can be visually quantified by the use of a fluorescence microscope. To study kinetics and concentration-response of drug-induced caspase-3 activation we exposed cells towards trans-beta-nitrostyrene, a rapidly acting experimental agent, as well as towards 5-fluorouracil, a standard agent with slow pro-apoptotic kinetics. RESULTS: Viability tests confirmed a comparable cytotoxic sensitivity of the transfected LoVo(EYFP) and the parental non-transfected cells towards trans-beta-nitrostyrene, a rapidly acting experimental agent, as well as towards 5-fluorouracil, a standard agent with slow kinetics. When comparing both caspase-3 assays at the same time points and concentrations of both agents, the new microscopic assay proved to be more sensitive, especially at lower concentrations and at earlier time points. DISCUSSION: Thus, visual detection of caspase activation in each affected cells enabled a more careful evaluation of the concentration- and time-dependence of drug-induced apoptosis which should also be useful with other experimental or standard agents or with other tumour cells.

Comparison of the rapid pro-apoptotic effect of trans-beta-nitrostyrenes with delayed apoptosis induced by the standard agent 5-fluorouracil in colon cancer cells.

Trans-beta-nitrostyrene (TBNS) has been reported to be a potent inhibitor of protein phosphatases PTB1 and PP2A and to display a pro-apoptotic effect even in multidrug resistant tumour cells. Here we compared the anti-tumour potential of TBNS with 5-fluorouracil (5-FU) as the standard chemotherapeutic agent for colorectal cancer in LoVo cells. Resistance to 5-FU based therapy might be a consequence of 5-FU's delayed effect requiring long-term effective concentrations in the tumour tissue. Thus, alternatives like platin containing drugs with a more rapid effect have been introduced recently. Compared to 5-FU TBNS displayed a faster cytotoxic and pro-apoptotic effect. A 50% decrease in viability was observed already after 8 h with TBNS while 5-FU displayed no significant effect before 48 h. DNA fragmentation and caspase-3 assays confirmed the more rapid apoptotic effect of TBNS. Since apoptosis affects individual cells these results about a rapidly induced apoptosis were further studied on a single cell level in microscopic assays of caspase-3 and caspase-8 activation. Adducts of trans-beta-nitrostyrene displayed an anti-tumour effect comparable to TBNS which suggests the possibility of creating adducts with optimised tissue targeting. Finally, the calculation of a drug combination index displayed a synergistic effect for the combination of TBNS and 5-FU in Lovo as well as in HT-29 and HCT116 colon cancer cells.

Toxicology and carcinogenesis studies of alpha-methylstyrene (Cas No. 98-83-9) in F344/N rats and B6C3F1 mice (inhalation studies).

UNLABELLED: alpha-Methylstyrene is used in the production of acrylonitrile-butadiene-styrene resins and copolymers, which improve the impact and heat-resistant properties of polymers, specialty grades of plastics, rubber, and protective coatings. alpha-Methylstyrene also moderates polymerization rates and improves product clarity in coatings and resins. Low molecular weight liquid polymers are used as plasticizers in paints, waxes, adhesives, and plastics. alpha-Methylstyrene was nominated by the U.S. Environmental Protection Agency for toxicologic evaluation and genotoxicity studies based on its high production volume and limited information available on its toxicity. Male and female F344/N rats and B6C3F1 mice were exposed to alpha-methylstyrene (99.5% pure) by inhalation for 3 months or 2 years. Inhalation studies were conducted because the primary route of human exposure is via inhalation. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were exposed by whole-body inhalation to alpha-methylstyrene at concentrations of 0, 75, 150, 300, 600, or 1,000 ppm for 6 hours per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Kidney weights were significantly increased in 1,000 ppm males and 600 and 1,000 ppm females. Statistically significant increases in liver weights occurred in 150 ppm or greater males and 600 and 1,000 ppm females. The incidences of renal hyaline droplet accumulation were similar between exposed groups and chamber control groups, but the severity of hyaline droplet accumulation in 600 and 1,000 ppm males was greater than in chamber controls. Consistent with the hyaline droplet accumulation, an exposure-related increase in alpha2µ-globulin was detected in the kidneys of males exposed to alpha-methylstyrene. Morphologic changes were not detected in the liver. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed by whole-body inhalation to alpha-methylstyrene at concentrations of 0, 75, 150, 300, 600, or 1,000 ppm for 6 hours per day, 5 days per week for 14 weeks. Two female mice in the 1,000 ppm group died before exposure on day 3. Final mean body weights of 600 and 1,000 ppm males and 75, 300, and 1,000 ppm females were significantly less than those of the chamber controls; final mean body weight gains of mice exposed to 300 ppm or greater were also significantly less. Moderate to severe sedation (males only) and ataxia were observed in 1,000 ppm mice. The absolute liver weights of 600 and 1,000 ppm females and the relative liver weights of 300, 600, and 1,000 ppm males and females were significantly increased. The estrous cycle lengths of 600 and 1,000 ppm female mice were significantly longer than that of the chamber controls. Minimal to mild centrilobular hypertrophy was present in the livers of male and female mice exposed to 600 or 1,000 ppm alpha-methylstyrene. The incidences of exposure-related nasal lesions, including atrophy and hyperplasia of Bowman's glands and atrophy and metaplasia of the olfactory epithelium, were significantly increased in all exposed groups of males and females. The incidences of hyaline degeneration, characterized by the accumulation of eosinophilic globules in the cytoplasm of the respiratory epithelium, were significantly increased in females exposed to 150 ppm or greater. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed by whole body inhalation to alpha-methylstyrene at concentrations of 0, 100, 300, or 1,000 ppm for 6 hours per day, 5 days per week, except holidays, for 105 weeks. Survival rates of exposed male and female rats were similar to those of the chamber controls. The mean body weights of 1,000 ppm males and females were less than those of the chamber control groups during year 2 of the study. Two 1,000 ppm males and one 300 ppm male had renal tubule carcinomas, and one 300 ppm male had a renal tubule adenoma. Because of the neoplasms observed in 300 and 1,000 ppm males at the end of the 2-year study and the finding of alpha2µ-globulin accumulation in the kidneys at 3 months, which is often associated with kidney neoplasms, additional step sections of kidney were prepared; additional males with focal hyperplasia or adenoma were identified. The incidences of renal tubule adenoma and carcinoma (combined) in the 1,000 ppm males were significantly greater than those in the chamber controls when the single and step sections were combined. The incidence of mineralization of the renal papilla was significantly increased in 1,000 ppm males. The incidence of mononuclear cell leukemia in 1,000 ppm males was significantly increased compared to the chamber controls. In the nose, the incidences of basal cell hyperplasia were significantly increased in all exposed groups of males and females, and the incidences of degeneration of the olfactory epithelium were increased in 1,000 ppm males and females and 300 ppm females. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed by whole body inhalation to alpha-methylstyrene at concentrations of 0, 100, 300, or 600 ppm for 6 hours per day, 5 days per week, except holidays, for 105 weeks. Survival of all exposed male and female mice was similar to that of the chamber control groups. Mean body weights of 600 ppm males were less than those of the chamber control group throughout the study, and those of 600 ppm females were less after week 13. The mean body weights of 300 ppm males and females were less than those of the chamber controls during much of the study, but these groups recovered by the end of the study. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in the 100 and 600 ppm males and in all exposed groups of females. The incidences of hepatocellular adenoma were significantly increased in all exposed groups of females, and the incidences in all exposed groups of males and females exceeded the historical range for chamber controls. The incidences of hepatocellular carcinoma and eosinophilic foci of the liver were significantly increased in 600 ppm females. The incidences of olfactory epithelial metaplasia and hyperplasia of the glands overlying the olfactory epithelium were significantly increased in all exposed groups of males and females. In addition, atrophy of the olfactory epithelium was significantly increased in 300 and 600 ppm males. The incidence and severity of nephropathy were increased in 600 ppm females compared to chamber controls. Epithelial hyperplasia of the forestomach also was present in male mice. GENETIC TOXICOLOGY: alpha-Methylstyrene was not mutagenic in four strains of Salmonella typhimurium, with or without rat or hamster liver metabolic activation enzymes (S9). alpha-Methylstyrene did not induce chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9 activation, but did significantly increase the frequency of sister chromatid exchanges in cultures exposed in the presence of S9. In vivo, no significant increases in the frequencies of micronucleated erythrocytes were seen in blood samples of male mice obtained at the conclusion of the 3-month study. However, in female mice from the 3-month study, a significant increase in micronucleated erythrocytes was observed in the 1,000 ppm group. CONCLUSIONS: Under the conditions of this 2-year inhalation study, there was some evidence of carcinogenic activity of alpha-methylstyrene in male F344/N rats based on increased incidences of renal tubule adenomas and carcinomas (combined). The increased incidence of mononuclear cell leukemia in 1,000 ppm male F344/N rats may have been related to alpha-methylstyrene exposure. There was no evidence of carcinogenic activity of alpha-methylstyrene in female F344/N rats exposed to 100, 300, or 1,000 ppm. There was equivocal evidence of carcinogenic activity of alpha-methylstyrene in male B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma or carcinoma (combined). There was clear evidence of carcinogenic activity of alpha-methylstyrene in female B6C3F1 mice based on increased incidences of hepatocellular adenomas and carcinomas. Exposure of rats to alpha-methylstyrene resulted in kidney toxicity, which in males exhibited some features of alpha2µ-globulin nephropathy. Exposure to alpha-methylstyrene resulted in nonneoplastic lesions of the nose in male and female rats and mice and of the liver and kidney in female mice.

Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges.

Styryl dyes have been among the most widely used probes for mapping membrane potential changes in excitable cells. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450-550 nm range. Longer wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering, improve recording from deep within tissue. In this paper we report on our efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra out to 900 nm. We have prepared and characterized dyes based on 47 variants of the styryl chromophores. Voltage-dependent spectral changes have been recorded for these dyes in a model lipid bilayer and from lobster nerves. The voltage sensitivities of the fluorescence of many of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because some of the dyes are often poorly water soluble, we have developed cyclodextrin complexes of the dyes to serve as efficient delivery vehicles. These dyes promise to enable new experimental paradigms for in vivo imaging of membrane potential.

[A case of diffuse invasive hepatocellular carcinoma associated with thrombosis of the main trunk of the portal vein in which hepatic transcatheter arterial chemoembolization concomitant to the use of degradable starch microspheres (DSM-TACE) was very effective].

The case reported here was a 80-year-old male who was presented with a diffuse type of hepatocellular carcinoma accompanied by thrombosis of the main trunk of the portal vein. The primary focus and portal vein thrombosis improved considerably following repeated transcatheter arterial chemoembolization using DSM (DSM-TACE). DSM-TACE was suggested to have the potential for becoming a new treatment method for advanced hepatocellular carcinoma, for which conventional transcatheter arterial embolization (TAE) using Lipiodol and so forth is contraindicated tue to thrombosis of the main trunk of the portal vein.

RISUG (reversible inhibition of sperm under guidance)--an antimicrobial as male vas deferens implant for HIV free semen.

HIV transmission from the male to the female is a major health problem. A hypothesis proposing an intra vas deferens implant of an antimicrobial compound to prevent the infection spread is presented. Mechanisms of action for the inhibition could include inactivating HIV in sperms passing through the vas deferens; drug release from the implant to destroy HIV entering into semen from genital structures distal to the vas deferens; and sperm acrosome released hyaluronidase mediated reabsorption of HIV. A subcomponent of the implant flowing along sperm pathway may have a role in reducing the entry of HIV from a positive female into penile tissue. A new drug RISUG (reversible inhibition of sperm under guidance) presently undergoing clinical trials for its contraceptive effect in the male (because it disrupts the sperm acrosome by an electrical charge and pH lowering effects) has also antimicrobial action. The drug being a combination of styrene maleic anhydride (SMA) and dimethyl sulfoxide (DMSO) on being injected into the lumen of the vas deferens produces styrene maleic acid thereby lowering pH; induces electrochemical action leading to a stable electrical charge generation; releases mandelic acid; and induces acrosome reaction in sperms with consequent release of hyaluronidase and sperm inactivation. Moreover, one time administration into the lumen of the vas gives long term action. All these phenomena very well match with the needs for HIV clearance of semen and hence RISUG is here proposed as a possible candidate material for the HIV inhibiting vas deferens implant when delivered in below contraceptive threshold dosage. For experimental validation, after obtaining data on the semen HIV load under control conditions in the HIV positive males inducted into the study, 30 mg of SMA in 120 microl of DMSO (contraceptive dose being 60 mg SMA+120 microl DMSO) is to be injected into vasa deferens bilaterally. Thereafter at intervals of one month the viral load needs to be determined in semen obtained either by masturbation or in lubricant free condom at intercourse - the method of collection remaining the same throughout for a particular subject. A significant reduction in the semen viral load following RISUG administration will validate the hypothesis. Speculated reduced female to male HIV transmission is more difficult to test. Nonspecific indications will come from a population study of the incidence of RISUG treated men becoming HIV positive as compared to that in the general population.

The chemotherapy of rodent malaria. LX. The importance of formulation in evaluating the blood schizontocidal activity of some endoperoxide antimalarials.

The activities of artemisinin (QHS) and a number of its semi-synthetic analogues, as well as Fenozan B07 (B07), a synthetic 1,2,4-trioxane, and arteflene (ATF), a synthetic surrogate of yingzhaosu, were compared in mice infected with drug-sensitive Plasmodium berghei or chloroquine-resistant P. yoelii ssp. NS. The studies were stimulated by the observation that B07, in certain aqueous preparations, appears to be equipotent by the subcutaneous (sc) or oral (po) routes in the rodent model but not in a simian model. In the rodent model, B07 was found to undergo rapid alteration (with a half-life of <24h) in an aqueous stock solution prepared using dimethyl sulphoxide (DMSO) to pre-dissolve the drug. Therefore, for all later experiments with aqueous preparations, the test material was newly formulated each day. In a carboxymethylcellulose formulation used as a 'standard suspending vehicle' (SSV), B07 and dihydroartemisinin (DIHYD) were found to be, respectively, one sixth and one 10th as active po as when the drugs were pre-dissolved in DMSO and then diluted with water. ATF in DMSO given po was less than one 20th as active as when used sc in the rodent model, and this drug in SSV was almost inactive po. The relatively low oral activity of these three compounds (especially DIHYD and ATF) may be attributable to extensive first-pass metabolism in the mouse. Oral beta-artemether (AM) and beta-arteether (AE) were highly active when used in SSV. ATF has been found to have low activity in simian models and clinical trials because of its poor absolute bio-availability. In in-vivo studies of the blood schizontocidal action of anti-malarials, in rodent malaria models, the data collected on the structure-activity relationships (SAR) of the drugs must be viewed critically when selecting specific compounds from a chemical series for further development. A study of the influence of drug formulation on the activity of other, novel antimalarials is crucial to the evaluation of the drugs, and merits high priority.

Effect of a new injectable male contraceptive on the seminal plasma amino acids studied by proton NMR spectroscopy.

Effect of RISUG, a newly developed male contraceptive, on various amino acids of seminal plasma ejaculates was studied by proton magnetic resonance spectroscopy at 400 MHz. Levels of amino acids were compared with the seminal plasma of obstructive azoospermia and controls. Glutamic acid, glutamine, and arginine were found to be high in concentration in human seminal plasma. The concentration of aromatic amino acids such as tyrosine, histidine, and phenylalanine in RISUG-injected subjects showed no significant difference compared to controls (p > 0.1); however, there was a statistically significant decrease in the concentration of these amino acids in obstructive azoospermia. The concentration of some prominent amino acids that showed overlapping resonances, such as isoleucine+leucine+valine (p < 0.01), alanine+isoleucine+lysine (p < 0.01), arginine+lysine+leucine (p < 0.01), and glutamic acid+glutamine (p < 0.01), showed a statistically significant decrease in RISUG-injected subjects compared to controls. Overlap of these amino acid resonances were noticed even at 600 MHz. In general, the total amino acids concentration in RISUG-injected subjects was found to be higher than in azoospermic subjects, confirming the occurrence of 'partial' obstructive azoospermia in subjects injected with this contraceptive.