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In the present work, we evaluated the antifungal activities of two novel ebselen analogs, N -allyl-benzisoselenazol-3(2H)-one ( N -allyl-bs) and N -3-methylbutylbenzisoselenazol-3(2H)-one ( N -3mb-bs). Colorimetric and turbidity assays were performed to determine the minimum inhibitory concentration (MIC) of these compounds in S1 (fluconazole-sensitive) and S2 (fluconazole-resistant) strains of C. albicans . N -3mb-bs was more active than the N -allyl-bs compound. It is noteworthy that the concentration of N -3mb-bs observed to inhibit fungal growth by 50% (18.2 µM) was similar to the concentration observed to inhibit the activity of the yeast plasma membrane H + -ATPase (Pma1p) by 50% (19.6 µM). We next implemented a mouse model of vulvovaginal candidiasis (VVC) using the S1 strain and examined the mouse and yeast proteins present in the vaginal lavage fluid using proteomics. The yeast proteins detected were predominately glycolytic enzymes or virulence factors associated with C. albicans while the mouse proteins present in the lavage fluid included eosinophil peroxidase, desmocollin-1, and gasdermin-A. We then utilized the N -3mb-bs compound (12.5 mg/kg) in the mouse VVC model and observed that it significantly reduced the vaginal fungal burden, histopathological changes in vagina tissue, and expression of myeloperoxidase (MPO). All in all, the present work has identified a potentially promising drug candidate for VVC treatment.

BRAF inhibitors (BRAFi) like vemurafenib (VEM) provide initial regression in mutated melanoma but rapidly develop resistance. Molecular pathways responsible for development of resistance against VEM finally converge towards the activation of oncogenic c-Myc. We identified an epigenetic approach to inhibit the c-Myc expression and resensitize BRAFi-resistant melanoma cells. ARV-825 (ARV) was employed as a BRD4 targeted PROteolysis TArgeting Chimera that selectively degrades the BRD4 to downregulate c-Myc. ARV synergistically enhanced the cytotoxicity of VEM in vitro to overcome its resistance in melanoma. Development of ARV and VEM-loaded lipid nanocomplex (NANOVB) significantly improved their physicochemical properties for oral delivery. Most importantly, oral administration of NANOVB substantially inhibited tumor growth at rate of 41.07 mm 3 /day in nude athymic mice. NANOVB treatment resulted in prolonged survival with 50% of mice surviving until the experimental endpoint. Histopathological analysis revealed significant tumor necrosis and downregulation of Ki-67 and BRD4 protein in vivo. Promising in vivo antitumor activity and prolonged survival demonstrated by NANOVB signifies its clinical translational potential for BRAFi-resistant melanoma., Competing Interests: Declaration of Competing Interest All authors have no competing interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In the present work, a series of N -terpenyl organoselenium compounds (CHB1-6) were evaluated for antimycotic activity by determining the minimum inhibitory concentration (MIC) for each compound in fluconazole (FLU)-sensitive (S1) and FLU-resistant (S2) strains of Candida albicans ( C. albicans ). The most active compounds in the MIC screen were CHB4 and CHB6, which were then evaluated for cytotoxicity in human cervical cancer cells (KB-3-1) and found to be selective for fungi. Next, CHB4 and CHB6 were investigated for skin irritation using a reconstructed 3D human epidermis and both compounds were considered safe to the epidermis. Using a mouse model of vulvovaginal candidiasis (VVC), CHB4 and CHB6 both exhibited antimycotic efficacy by reducing yeast colonization of the vaginal tract, alleviating injury to the vaginal mucosa, and decreasing the abundance of myeloperoxidase (MPO) expression in the tissue, indicating a reduced inflammatory response. In conclusion, CHB4 and CHB6 demonstrate antifungal activity in vitro and in the mouse model of VVC and represent two new promising antifungal agents., Competing Interests: The authors declare no conflict of interest.

Background: Vulvovaginal candidiasis is primarily caused by Candida albicans ( C. albicans ). Here, a novel organoselenium compound (G20) was synthesized and evaluated for anti- Candida activity. Methods: Growth-inhibition studies and medium acidification assays to assess the inhibition of the yeast plasma membrane H + -ATPase (Pma1p) were carried out in vitro using G20. A self-nanoemulsifying formulation (SNEP) of G20 was prepared and evaluated for antimycotic activity in a mouse model. Results: G20 inhibited the growth of C. albicans through a mechanism that, at least in part, involves the inhibition of Pma1p. The G20-SNEP formulation significantly reduced vaginal colonization and vaginal inflammation relative to yeast-infected but untreated control mice. Conclusion: G20-SNEP exhibits potent antimycotic activity in a mouse model of vulvovaginal candidiasis.

Mechlorethamine (HN2) is a derivative of the chemical warfare agent sulfur mustard (SM) and cutaneous exposure to HN2 is associated with dermal-epidermal junction (DEJ) disruption (vesication). The primary purpose of the present study was to investigate the effect of HN2 on the mammalian target of rapamycin (mTOR) signaling pathway using an in vivo mouse ear vesicant model (MEVM). To this end, the ears of male C57BL/ 6 J mice were exposed to a single topical dose of HN2 (100 mM) or vehicle control (DMSO). Mice were then euthanized 30 min, 1 h or 24 h following exposure. Mouse ear skin exposed to HN2 and biopsied 24 h thereafter exhibited increased tissue expression of Raptor, an important member of the mTORC1 complex, relative to vehicle treated samples. HN2 reduced the downstream effectors phospho S6 (Ser 240/244) ribosomal protein and phospho 4E-BP1 (Thr 37/46) of the mTOR pathway in the epidermis at 30 min, 1 h and 24 h following HN2 exposure but not in the dermis. These results support the hypothesis that HN2-mediated cutaneous toxicity involves dysregulation of the mTOR signaling pathway in the epidermis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The purpose of the present study was to investigate the vesicant countermeasure effects of hydrocortisone (HC) and ebselen (EB-1), administered as monotherapy or as a combination treatment. The mouse ear vesicant model (MEVM) was utilized and test doses of HC (0.016, 0.023, 0.031, 0.047, 0.063, 0.125 or 0.250 mg/ear), EB-1 (0.125, 0.187, 0.250, 0.375 or 0.500 mg/ear) or the combination of HC + EB-1 were topically applied at 15 min, 4 h and 8 h after nitrogen mustard exposure. Ear punch biopsies were obtained 24 h after mechlorethamine (HN2) exposure. Compared to control ears, ear tissues exposed topically to HN2 (0.500 µmol/ear) presented with an increase in ear thickness, vesication, TUNEL fluorescence and expression of matrix metalloproteinase 9 (MMP-9) and inducible nitric oxide synthase (iNOS). In contrast, HN2 exposed ears treated topically with EB-1 showed a significant decrease in morphometric thickness and vesication vs. HN2 alone. Ear tissues exposed to HN2 and then treated with HC also demonstrated reductions in morphometric thickness and vesication. Combination treatment of HC + EB-1 was found to be the most effective at reducing HN2-induced ear edema and vesication. The combination also dramatically decreased HN2-mediated cutaneous expression of iNOS and MMP-9 and decreased HN2-induced TUNEL staining. Taken together, our study demonstrates that the combination of HC + EB-1 is an efficacious countermeasure to HN2., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Vulvovaginal candidiasis (VVC), caused by Candida albicans, is a common infection in women affecting their quality of life. Standard antifungal drugs (e.g., fluconazole, itraconazole) are typically fungistatic or rendered ineffective due to drug resistance indicating an urgent need to build an arsenal of novel antifungal agents. To surmount this issue, we tested the hypothesis that the organoselenium compound ebselen (EB) possesses antifungal efficacy in a mouse model of VVC. EB is a poorly water-soluble drug and DMSO as a vehicle has the potential to exhibit cytotoxic effects when administered in vivo. EB loaded self-nanoemulsifying preconcentrate (EB-SNEP) was developed, characterized in vitro, and tested in a mouse model of VVC. In vivo studies carried out with EB-SNEP (12.5 mg/kg) showed a remarkable decrease in infection by ~562-fold compared to control (infected, untreated animals). Taken together, EB nanoemulsion proved to be an effective and promising antifungal agent., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Carvacrol (CAR), a phenolic monoterpenoid, has been extensively investigated for its antimicrobial and antifungal activity. As a result of its poor physicochemical properties, water soluble carvacrol prodrugs (WSCPs) with improved water solubility were previously synthesized and found to possess antimicrobial activity. Here, three novel CAR analogs, WSCP1, WSCP2, and WSCP3, were tested against fluconazole (FLU)-sensitive and -resistant strains where they showed greater antifungal activity than CAR against C. albicans . The probable mechanism by which the CAR prodrugs exert the antifungal activity was studied. Results from medium acidification assays demonstrated that the CAR and its synthetically designed prodrugs inhibit the yeast plasma membrane H+-ATPase (Pma1p), an essential target in fungi. In other words, in vitro data indicated that CAR analogs can prove to be a better alternative to CAR considering their improved water solubility. In addition, CAR and WSCP1 were developed into intravaginal formulations and administered at test doses of 50 mg/kg in a mouse model of vulvovaginal candidiasis (VVC). Whereas the CAR and WSCP1 formulations both exhibited antifungal efficacy in the mouse model of VVC, the WSCP1 formulation was superior to CAR, showing a remarkable decrease in infection by ~120-fold compared to the control (infected, untreated animals). Taken together, a synthetically designed prodrug of CAR, namely WSCP1, proved to be a possible solution for poorly water-soluble drugs, an inhibitor of an essential yeast pump in vitro and an effective and promising antifungal agent in vivo.

Epidemiological findings have discussed recurrent and persistent vulvovaginal candidiasis to be a major manifestation of HIV infected women. Conversely, women with vulvovaginal candidiasis have higher risk of acquiring HIV transmitted during intercourse. Common treatments for such conditions include combined antiretroviral and antifungal therapy. Drug-Drug interaction is a major problem encountered due to common CYP450 metabolic pathway of azoles and antiretroviral drugs. Ebselen (EB), lipophilic, organo-selenium compound has demonstrated promising anti-HIV and anti-fungal activity. The aim of current research was to develop and characterize a rapidly soluble and non-cytotoxic vaginal film of ebselen which could serve dual purpose of treating vulvovaginal candidiasis and pre-exposure prophylactic (PrEP) against HIV. Ebselen/cyclodextrin polymer/Soluplus® (1:10:10) ternary complex (EβpolySol) showed 200 fold enhancement in aqueous solubility and no degradation of EB in thermogravimetry analysis. EβpolySol film with tensile strength of 33.12 ± 1.98 N/cm 2 disintegrated within 30 sec, presented instant drug release with no apparent precipitation in simulated vaginal fluid. EβpolySol film showed compatibility with HEC-1A monolayer and HeLa cells compared to VCF®. EβpolySol film showed MIC of 20 μM against Candida species and IC 50 of 0.71 μM against HIV., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard mimetic. Topical exposure to HN2 is associated with tissue blistering. Previous work in our laboratory has shown that ebselen (EB-1) possesses anti-vesicant, anti-inflammatory, anti-bacterial, anti-fungal, and cytoprotective properties, both in vivo and in vitro . We recently reported that ebselen oxide (EB-2), an analog of EB-1 with a tetravalent selenium atom, also possesses anti-bacterial and anti-fungal activity and confers cytoprotection against HN2 in vitro . The purpose of the present study was to determine the vesicant countermeasure potential of EB-2 using the mouse ear vesicant model (MEVM). Compared to control ears, mouse ears exposed to a single dose of HN2 (0.500 µmol/ear) showed an increase in wet weights, ear thickness, hyperplasia, vesication, and inflammatory cell infiltration after 24 h. Fluorescence microscopy of terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-stained sections showed that the occurrence of apoptosis extended from the epidermis of the HN2-treated side, all the way to the contralateral epidermis. In contrast, HN2-exposed ears treated topically with EB-2 at a test dose of 0.250 mg/ear showed a significant decrease in wet weight (12% less vs. HN2 alone), morphometric thickness (13% less vs. HN2 alone), and vesication. In addition, TUNEL staining revealed that HN2 ears treated with EB-2 (0.250 mg/ear) showed a decrease in apoptosis as compared to the HN2 group. EB-2 also reduced the abundance of matrix metalloproteinase-9 (MMP-9) in ear tissues exposed to HN2. Taken together, our study demonstrates that EB-2 is an efficacious countermeasure to HN2.

Superficial mycoses are the fungal infections of skin, hair and nail which affect thousands of people worldwide. Emerging resistance to azole antifungals is a common problem in the treatment of superficial or systemic fungal infection. Ebselen (EB), an organoselenium compound, has demonstrated promising activity against pathogenic yeasts. EB showed negligible dynamic and kinetic solubility in water (~ 4.2 µg/mL) which severely limits the scope of conventional formulations. The objective of present study was to develop and characterize a novel topical nanoemulgel of EB for enhancing solubility and permeability. Based on saturation solubility study, EB loaded self-nanoemulsifying preconcentrate (EB-P) was prepared using Dimethylacetamide, Kolliphor® ELP and Medium chain triglyceride which spontaneously formed 54.82 ± 1.26 nm size nanoglobules with zeta potential of -1.69 mV. Nanoemulgel was prepared by homogenous dispersion of EB-P in various gel/ointment bases. Scanning electron microscopy images showed significant drug precipitation in nanoemulgels prepared without Soluplus®. Rheological study confirmed shear thinning behavior of Soluplus® based HPMC K4M (SBH) gel. EB-P loaded SBH showed 2.3 and 5-fold higher Strat-M® deposition of EB compared to HPMC gel and Aquaphor®, respectively. EB-P showed marked anti-fungal activity at 20 µM against Candida albicans and Candida tropicalis while terbinafine was ineffective even at 100 µM concentration. Thus, topical nanoemulgel of EB could be a promising alternative to existing therapy for treatment of candidiasis., Competing Interests: Declaration of Competing Interest All authors have no competing interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 μmol/ear) or DMSO vehicle (5 μl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures., (Copyright © 2018 SETOX & Institute of Experimental Pharmacology and Toxicology, SASc.)

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H + -ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)-resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC 50  ∼ 18 μM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC 50  ∼ 14 μM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU-resistant fungi., (© 2017 Wiley Periodicals, Inc.)

The present study investigated the pharmacological effects of three stilbenoids, resveratrol (RES), pterostilbene (PTR) and piceatannol (PIC), in transformed and normal macrophages. Our first aim was to comparatively assess the cytotoxicity of RES, PTR and PIC in unstimulated transformed mouse macrophages (RAW 264.7 cells) and primary peritoneal macrophages (PMs) harvested from both wild type and Nrf2 (nuclear factor erythroid 2-related factor 2)-deficient female mice. Our second aim was to investigate whether the inhibitory effect of RES, PTR and PIC on nitric oxide (NO) release from stimulated PMs depends on the status of the transcription factor Nrf2. The rationale for investigating Nrf2 status was based upon recent reports showing that certain compounds (sulforaphane and linalool) suppress LPS-induced inflammation in an Nrf2-dependent manner. Cell viability studies confirmed our prior work in unstimulated RAW 264.7 cells, with cytotoxic potency decreasing in the order of PTR > PIC > RES. Unstimulated PMs, regardless of Nrf2 status, were less sensitive to stilbenes, requiring at least a threefold higher stilbene concentration to inhibit cell viability, with cytotoxic potency again decreasing in the order of PTR > PIC > RES. In studies focused on our second aim, IC 50 values for NO inhibition (measured as [Formula: see text]) in wild type PMs were similar for all three stilbenes (∼10 μM). In Nrf2-deficient PMs, the IC 50 for NO inhibition by PIC did not change; however, a rightward shift in the concentration effect curve was observed for both RES and PTR, indicating a role for Nrf2 in the suppression of LPS-induced [Formula: see text] accumulation by these particular stilbenes.

Resveratrol and pterostilbene are natural products that are present in plants and have been incorporated into various dietary supplements. Numerous beneficial pharmacologic effects have been reported for these stilbenes; however, the mechanism by which these compounds exert a cytotoxic effect in RAW 264.7 macrophages has not been well characterized. We have previously described that resveratrol is toxic to these tumor-derived macrophages and that stimulation with lipopolysaccharide (LPS) reduces resveratrol toxicity via a mechanism that involves activation of toll like receptor 4. In the present work, we examined the cellular and molecular effects of resveratrol and the related compound pterostilbene by determining cell viability and caspase 3 activity in control and LPS-stimulated RAW 264.7 macrophages incubated with these stilbenes for 24 h. We found that LPS stimulation reduced the cytotoxicity of resveratrol but not of pterostilbene in these cells. When examined for effects on caspase 3 activation after a 24 h incubation, resveratrol and pterostilbene were each found to separately and significantly increase caspase 3 activity in these cells. LPS stimulation prevented caspase 3 activation by pterostilbene and reduced caspase 3 activation by resveratrol in RAW 264.7 macrophages. The data presented here indicate that LPS induces a phenotype switch in tumor-derived RAW 264.7 macrophages in which cells experiencing LPS in the presence of resveratrol or pterostilbene become less likely to activate the pro-apoptotic factor caspase 3.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Previous studies in this and other laboratories have demonstrated that ebselen (EB-1), an organoselenium compound, spares cells from mechlorethamine (HN2) toxicity in vitro. In the present study, the hypothesis that EB-1 will reduce dermal toxicity of HN2 in vivo is put forward and found to have merit. Using the mouse ear vesicant model (MEVM), HN2, applied topically, showed a dose-dependent effect upon ear swelling and thickness 24 h after treatment; whereas tissue injury consistent with vesication was observed at the higher test doses of HN2 (≥ 0.250 µmol per ear). To examine HN2 countermeasure activity using the MEVM, either hydrocortisone (HC), as a positive control, or EB-1, the test countermeasure, was administered as three topical treatments 15 min, 4 and 8 h after HN2 exposure. Using this approach, both HC and EB-1 were found to reduce tissue swelling associated with HN2 toxicity 24 h after exposure to the vesicant. Taken together, these data demonstrate for the first time the effectiveness of EB-1 as a vesicant countermeasure in a relevant in vivo model., (Copyright © 2013 John Wiley & Sons, Ltd.)

Purpose: To develop a live oral delivery system of Glucagon like peptide-1 (GLP-1), for the treatment of Type-2 Diabetes., Methods: LL-pUBGLP-1, a recombinant Lactococcus lactis (L. lactis)) transformed with a plasmid vector encoding GLP-1 cDNA was constructed and was used as a delivery system. Secretion of rGLP-1 from LL-pUBGLP-1 was characterized by ELISA. The bioactivity of the rGLP-1 was examined for its insulinotropic activity on HIT-T15 cells. Transport of rGLP-1 across MDCK cell monolayer when delivered by LL-pUBGLP-1 was studied. The therapeutic effect of LL-pUBGLP-1 after oral administration was investigated in ZDF rats., Results: DNA sequencing and ELISA confirmed the successful construction of the LL-pUBGLP-1 and secretion of the active form of rGLP-1. In vitro insulinotropic studies demonstrated that LL-pUBGLP-1 could significantly (p < 0.05) stimulate HIT-T15 cells to secrete insulin as compared to the controls. When delivered by LL-pUBGLP-1, the GLP-1 transport rate across the MDCK cell monolayer was increased by eight times (p < 0.01) as compared to the free solution form. Oral administration of LL-pUBGLP-1 in ZDF rats resulted in a significant decrease (10-20%, p < 0.05) in blood glucose levels during 2-11 h post dosing and a significant increase in insulin AUC0-11h (2.5 times, p < 0.01) as compared to the free solution., Conclusion: The present study demonstrates that L. lactis when genetically modified with a recombinant plasmid can be used for the oral delivery of GLP-1.

Sulfur mustard (SM) is a potent vesicant. The lack of an effective antidote makes SM a continued threat to both military and civilian settings. A surrogate agent, namely mechlorethamine (HN2), was used here to mimic the toxicity of SM, and the main objective of this study was to demonstrate if selected organoselenium analogs could protect cultured A-431 skin cells from HN2 toxicity. Test compounds included ebselen (EB-1) and three related organoselenium analogs (EB-2, EB-3 and EB-4). In the absence of test compound, a reproducible and robust cell death was observed in the cells following incubation with HN2 (25 µM, 24 or 48 h) while cells treated with test compound alone (15, 30 or 60 µM) for similar periods of time were generally not affected. When incubated in the presence of both HN2 and test compound for 24 or 48 h, it was found that EB-1, EB-2, EB-3 and EB-4 could spare the cells from death, with the EB-4 compound being the most effective at reducing HN2 toxicity. Light microscopy confirmed these findings. The organoseleniums were also examined for their effects on reducing lipid peroxidation in the A-431 skin cells. Among the test compounds, EB-4 reduced lipid peroxidation by HN2 to the greatest extent. These studies, taken together, validate that the organoselenium antioxidants tested here may serve a purpose in the discovery of medical countermeasures to vesicants.

A series of test compounds were evaluated for an ability to reduce the toxicity of the nitrogen mustard mechlorethamine (HN2) in vitro. The test compounds included resveratrol, pterostilbene, vitamin C, ebselen, ebselen diselenide, and ebselen-sulfur. Among them, ebselen demonstrated the highest degree of protection against HN2 toxicity. To this end, pretreatment of the cells with ebselen offered protection against the toxicant whereas no protection was observed when cells were first incubated with HN2 and then treated with ebselen. Significant increases in caspase 3 and caspase 9 activities were observed in response to HN2, and ebselen was found to reduce these effects. Taken together, the data presented here indicate that ebselen is an effective countermeasure to nitrogen mustard in vitro, which is worthy of future investigation in vivo., (© 2013 Wiley Periodicals, Inc.)

Vesicants are potent blistering agents. The prototype vesicant is sulphur mustard gas, first used in World War I, which still has no effective antidote. We used a mustard gas surrogate 2-chloroethyl ethyl sulphide (CEES) to study the ability of resveratrol (RES) and pterostilbene (PTS), two well-established stilbene antioxidants, ebselen (EB-1), an organoselenium compound, and three EB-1 analogues (EB-2, EB-3, and EB-4) to reduce CEES toxicity in human epidermoid carcinoma cells (A-431). Following a 24-hour incubation of a toxic concentration of CEES (1000 μmol L-1), we used the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide] test to analyse cell viability. Different concentrations of test antioxidants alone (15 μmol L-1, 30 μmol L-1 or 60 μmol L-1) did not decrease cell viability. Treatment with CEES and test antioxidants for 24 h showed that only EB-1 and its analogues EB-2, EB-3, and EB-4 but not the stilbene compounds could rescue the cells from death. EB-1 and EB-4 were the most effective at reducing CEES cytotoxicity and did so in a concentration-dependent manner, while EB-2 and EB-3 demonstrated the least protective effect. In summary, the data described herein indicate that organoselenium antioxidants, especially EB-4, may prove useful as countermeasures to blistering agents.

Introduction: Numerous studies employing various animal models have found that perinatal stress, encountered in utero during sensitive developmental stages or shortly after birth, disrupts both sexual differentiation and sexual behavior in offspring. The biochemical, cellular, genetic and epigenetic events which are involved in the organismal response to perinatal stress are currently under investigation., Methods, Results and Discussion: In this review, the reader is introduced to perinatal stressors as a toxicological phenomenon, and several recently characterized epigenetic responses to said stressors are discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A group of novel N-1-substituted indazole-3-carboxamide derivatives were synthesized and evaluated as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1). A structure-based design strategy was applied to a weakly active unsubstituted 1H-indazole-3-carboxamide 2, by introducing a three carbon linker between 1H-indazole-3-carboxamide and different heterocycles, and led to compounds 4 [1-(3-(piperidine-1-yl)propyl)-1H-indazole-3-carboxamide, IC(50) =36μm] and 5 [1-(3-(2,3-dioxoindolin-1-yl)propyl)-1H-indazole-3-carboxamide, IC(50) = 6.8μm]. Compound 5 was evaluated in rats for its protective action against diabetes induced by a treatment with streptozotocin, a known diabetogenic agent. In addition to preserving the ability of the pancreas to secrete insulin, compound 5 was also able to attenuate the ensuing hyperglycemic response to a significant extent., (© 2011 John Wiley & Sons A/S.)

The plasma membrane H(+)-ATPase pump (Pma1p) has been proposed as a viable target for antifungal drugs since this high capacity proton pump plays a critical role in the intracellular regulation of pH and in nutrient uptake of yeast and other fungi. In recent years, this and other laboratories have verified that the antifungal activity of 2-phenylbenzisoselenazol-3(2H)-one, an organoselenium compound commonly referred to as ebselen (1), stems, at least in part, from its inhibitory action on the fungal Pma1p. In the present study, the antifungal efficacy of 2-(3-pyridinyl)-benzisoselenazol-3(2H)-one (2) and 2-phenylbenzisoselenazol-3(2H)-one 1-oxide (3), two ebselen analogs, was evaluated using a strain of S. cerevisiae and compared against that of 1. In addition, the study also examined the inhibitory potential of these three compounds toward the Pma1p of S. cerevisiae. Based on mean IC(50) values, the antifungal potency was found to decrease in the order 3 > 1 > 2. However, in terms of inhibitory action on Pma1p, the potency decreased in the order 1 > 3 > 2. The magnitude of these activities appears to be correlated with the corresponding log P values, with compound 2 being the most hydrophilic and the least active of the three.

In an effort to find inhibitors that are effective against both Candida and Aspergillus spp., a series of 5(6)-(un)substituted benzotriazole analogs, represented by compounds 3a-3h and 3b'-3f', were prepared using a crystalline oxirane intermediate 1 previously synthesized in our laboratory. All the compounds were evaluated for inhibitory activity against various species of Candida and Aspergillus. Compounds 3b' (5,6-dimethylbenzotriazol-2-yl derivative), 3d (5-chlorobenzotriazol-1-yl derivative) and 3e' (6-methylbenzotriazol-1-yl derivative) exhibited potent antifungal activity, with the MICs for Candida spp. and Aspergillus niger, ranging from 1.6 microg/mL to 25 microg/mL and 12.5 microg/mL to 25 microg/mL, respectively. The present work describes the design, synthesis, regioisomer characterization (through COSY and NOESY 2D-NMR spectroscopy and single molecule X-ray crystallography), antifungal evaluation, molecular docking, and structure-activity relationships of the various 5(6)-(un)substituted benzotriazole analogs., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1-S and persistence of BRCA1 during G2/M. After gamma-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.

PMA1 is a yeast gene that codes for the plasma membrane H(+)-ATPase, a protein commonly referred to as Pma1p. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a synthetic selenium-containing compound that has recently been shown to display antimicrobial activity owing to its ability to inhibit Pma1p. Ebselen is able to block the activity of Pma1p not only in opportunistic pathogens such as Cryptococcus neoformans and Candida albicans but also in nonpathogenic yeasts such as Saccharomyces cerevisiae. A series of in vitro studies aimed at evaluating the antifungal activity of ebselen were performed. At low concentrations (<10 microM), ebselen was fungistatic against three strains of S. cerevisiae (IC(50) approximately 3 microM) and one fluconazole-resistant strain of C. albicans (IC(50) approximately 6 microM), and at a high concentration (30 microM) it was fungicidal against C. albicans. Moreover, ebselen was found to inhibit medium acidification by the fluconazole-resistant strain of C. albicans in a concentration-dependent manner. In comparison to currently used antifungal agents represented by azole (itraconazole, ketoconazole, fluconazole) and polyene (amphotericin B) compounds, ebselen was at least 10-fold more potent than fluconazole but less active than the other compounds tested. The present results suggest that the growth inhibitory activity of ebselen toward fluconazole-resistant yeast cells is due, at least in part, to inhibition of Pma1p. Ebselen may also serve as a useful agent in the treatment of infections caused by fluconazole-resistant fungi.

Germline mutations that inactivate BRCA1 are responsible for breast and ovarian cancer susceptibility. One possible outcome of genetic testing for BRCA1 is the finding of a genetic variant of uncertain significance for which there is no information regarding its cancer association. This outcome leads to problems in risk assessment, counseling and preventive care. The purpose of the present study was to functionally evaluate seven unclassified variants of BRCA1 including a genomic deletion that leads to the in-frame loss of exons 16/17 (Delta exons 16/17) in the mRNA, an insertion that leads to a frameshift and an extended carboxy-terminus (5673insC), and five missense variants (K1487R, S1613C, M1652I, Q1826H and V1833M). We analyzed the variants using a functional assay based on the transcription activation property of BRCA1 combined with supervised learning computational models. Functional analysis indicated that variants S1613C, Q1826H, and M1652I are likely to be neutral, whereas variants V1833M, Delta exons 16/17, and 5673insC are likely to represent deleterious variants. In agreement with the functional analysis, the results of the computational analysis also indicated that the latter three variants are likely to be deleterious. Taken together, a combined approach of functional and bioinformatics analysis, plus structural modeling, can be utilized to obtain valuable information pertaining to the effect of a rare variant on the structure and function of BRCA1. Such information can, in turn, aid in the classification of BRCA1 variants for which there is a lack of genetic information needed to provide reliable risk assessment.

The present work has evaluated the role of lipopolysaccharide (LPS) stimulation and of TLR4 receptors on the cytotoxicity of resveratrol (RES), a polyphenolic stilbene component of red wine, in TLR4-bearing (i.e., RAW 264.7) and TLR4-deficient (i.e., 10ScNCr/23) macrophages. Based on the results of the MTT assay, DNA fragmentation analysis, and scanning electron microscopic examination, cell stimulation with LPS was found to maintain the viability of, attenuate DNA fragmentation in, and preserve normal morphology of TLR4-proficient macrophages exposed to RES. In contrast, LPS failed to spare TLR4-deficient macrophages from the deleterious effects of RES. Moreover, while LPS treatment conferred protection from RES toxicity in TLR4-bearing macrophages, this stilbene inhibited the production of nitric oxide by stimulated cells in a concentration-dependent fashion. It is therefore likely that the cytotoxic effects of RES towards non-stimulated macrophages and the propensity of RES to inhibit nitric oxide production by activated macrophages are both contributing, at least in part, to the anti-inflammatory activity of this natural product.

Resveratrol (RES), a component of red wine, possesses anti-inflammatory properties. The studies described in the present work were aimed at evaluating the potential for RES and related stilbene analogs (piceatannol, PIC; pterostilbene, TPS; trans-stilbene, TS; and trans-stilbene oxide, TSO) to exhibit toxicity towards RAW 264.7 mouse macrophages. The effect of TS, TSO, RES and TPS on RAW 264.7 macrophage viability was determined by two standard methods: (a) the MTT assay and (b) the trypan blue dye exclusion test. Whereas macrophages were more sensitive to PIC (LC(50 trypan) approximately 1.3 muM) and to TPS (LC(50 trypan) approximately 4.0 microM and LC(50 MTT) approximately 8.3 microM) than to RES (LC(50 trypan) approximately 8.9 microM and LC(50 MTT) approximately 29.0 microM), they were relatively resistant to TSO (LC(50 trypan) approximately 61.0 microM and LC(50 MTT) > 100 microM) and to TS (LC(50 trypan) > or = 5.0 microM and LC(50 MTT) > or = 5.0 microM). The ability of selected stilbenes (RES, TPS and PIC) to exhibit growth inhibitory effects was also examined. Although RES and TPS were observed to inhibit cell proliferation in macrophages (IC(50) < or = 25 microM), these cells were resistant to growth inhibition by PIC (IC(50) > or = 50 microM). The data obtained in the present analysis demonstrate that substituted stilbene compounds such as RES have the capacity to exhibit cytotoxic and anti-proliferative activities in macrophages.

Mechlorethamine (HN2) and 2-chloroethylethyl sulfide (CEES) are mustard vesicants. Butylated hydroxyanisole (BHA) is a phenolic antioxidant. In the present work, the ability of BHA to reduce the toxicity of HN2 and CEES was investigated using A-431 skin cells. HN2 toxicity was found to be dependent, at least in part, on the cellular glutathione (GSH) status. BHA also decreased HN2-induced DNA damage and lipid peroxidation. The cytoprotective activity of BHA was also compared with that of the structurally unrelated antioxidant ebselen. Whereas ebselen (30 microM) protected skin cells from the toxicity of both HN2 (10-80 microM) and CEES (10-40 microM), BHA (100 microM) reduced the toxicity of HN2 only. Taken together, these data suggest that antioxidants such as BHA or ebselen may serve as useful treatments for injury caused by blistering agents.

The cytotoxicity of resveratrol and of piceatannol, a structural analog of resveratrol, was examined in cultured cells. Using a MTT-based assay, which measures the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to a colored formazan product in living cells, resveratrol was found to inhibit the viability of transformed mouse macrophages, tumor-derived human T cells and human epidermoid carcinoma cells in a concentration-dependent manner, with the effect decreasing in the order: T cells (LC50 approximately 27 micromol L(-1), 24 h; approximately 9 micromol L(-1); 48 h)>macrophages (LC50 approximately 29 micromol L(-1), 24 h; 39 micromol L(-1), 48 h)>skin cells (LC50 approximately 91 micromol L(-1), 24 h; approximately 66 micromol L(-1), 48 h). Paradoxically, a high concentration of resveratrol (50 micromol L(-1)) inhibited the proliferation of all three cell types, and a low concentration (5 micromol L(-1)) stimulated the proliferation of macrophages. The viability of macrophages was also decreased by piceatannol in a concentration-dependent manner. The stimulation of macrophages with zymosan lowered the cytotoxicity of both resveratrol and piceatannol. Scanning electron microscopy of cells treated with resveratrol revealed changes in cellular morphology that were consistent with toxicity. In macrophages and skin cells, resveratrol (50 micromol L(-1)) induced a time-dependent increase in reduced glutathione levels but did not alter the background levels of thiobarbituric acid-reactive substances. Taken together, the present data indicate that resveratrol is toxic to cultured macrophages, T cells and skin cells at concentrations>or=25 micromol L(-1), and that the cytotoxicity occurs via a mechanism that does not involve oxidative stress. Furthermore, the degree of toxicity of both resveratrol and piceatannol towards macrophages depends on the activation status of these cells, with zymosan-activated cells appearing more resistant than nonstimulated cells.

Nitrogen mustards are vesicants capable of burning the skin, eyes and respiratory tract of exposed individuals. While generally less toxic than sulfur mustards, these compounds have the potential for use as chemical warfare agents. Presently, no antidote exists for treatment against nitrogen mustard toxicity. The purpose of this study was to investigate the in vitro toxicity of the nitrogen mustard mechlorethamine (HN2) in four cell models: CEM-SS human T cells, A431 human skin epithelial cells, rat hippocampal astrocytes and rat pleural mesothelial cells. Furthermore, the efficacy of the synthetic seleno-organic compound ebselen (Eb) (2-phenyl-1,2- benzisoselenazol-3(2H)-one) as a cytoprotective agent against such toxicity was evaluated. Significant increases in cell viability, as assessed using an MTT assay for viability, was demonstrated when 30 microM Eb was used as a cotreatment with HN2 in all cell models tested at the following doses of HN2: A431 skin cells,10-40 microM; rat astrocytes, 20 and 40 microM; rat mesothelia, 10-40 microM; and human T cells 4-16 microM. Decreases in cell viability and toxicity to HN2 were confirmed using light and scanning electron microscopy. Membrane damage, observed with HN2 exposure, such as blebbing and loss of cell projections, was ameliorated with Eb cotreatment. Our results demonstrate a generalized protective effect observed with Eb cotreatment that suggests that this agent may have potential as an antidote for HN2 exposure and toxicity.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing antioxidant demonstrating anti-inflammatory and cytoprotective properties in mammalian cells and cytotoxicity in lower organisms. The mechanism underlying the antimicrobial activity of ebselen remains unclear. It has recently been proposed that, in lower organisms like yeast, the plasma membrane H+-ATPase (Pma1p) could serve as a potential target for this synthetic organoselenium compound. Using yeast and bacteria, the present study found ebselen to inhibit microbial growth in a concentration- and time-dependent manner, and yeast and Gram-positive bacteria to be more sensitive to this action (IC50 approximately 2-5 microM) than Gram-negative bacteria (IC50 < 80 microM). Washout experiments and scanning electron microscopic analysis revealed ebselen to possess fungicidal activity. In addition, ebselen was found to inhibit medium acidification by PMA1-proficient haploid yeast in a concentration-dependent manner. Additional studies comparing PMA1 (+/-) and PMA1 (+/+) diploid yeast cells revealed the mutant to be more sensitive to treatment with ebselen than the wild type. Ebselen also inhibited the ATPase activity of Pma1p from S. cerevisiae in a concentration-dependent manner. The interaction of ebselen with the sulfhydryl-containing compounds L-cysteine and reduced glutathione resulted in the complete and partial prevention, respectively, of the inhibition of Pma1p ATPase activity by ebselen. Taken together, these results suggest that the fungicidal action of ebselen is due, at least in part, to interference with both the proton-translocating function and the ATPase activity of the plasma membrane H+-ATPase., ((c) 2007 Wiley Periodicals, Inc.)

Macrophages play an important role in host-defense and inflammation. In response to an immune challenge, macrophages become activated and produce proinflammatory mediators that contribute to nonspecific immunity. The mediators released by activated macrophages include: superoxide anion; reactive nitrogen intermediates, such as nitric oxide and peroxynitrite; bioactive lipids; and cytokines. Although essential to the immune response, overproduction of certain macrophage-derived mediators during an immune challenge or inflammatory response can result in tissue injury and cellular death. The present report is focused on understanding some of the molecular mechanisms used by macrophages to produce reactive nitrogen intermediates in response to immunostimulatory agents such as heat shock protein 60 and bacterial lipopolysaccharide. The role of Toll-like receptors and transcription factors such as nuclear factor kappa B (NFkappaB) in the innate immune response is also described. A basic understanding of the underlying molecular mechanisms responsible for macrophage activation should serve as a foundation for novel drug development aimed at modulating macrophage activity.

Women carrying one mutated BRCA1 allele are at increased risk of developing breast and ovarian cancer but tumor initiation requires the loss of the wild-type allele indicating that it is a tumor suppressor gene. In the 10 years since the cloning of BRCA1, a function for the gene product in the DNA damage response has been established. However, identifying the exact biochemical activities of BRCA1 has been a more difficult task. Our current understanding suggests that the molecular functions mediated by the terminal ends of BRCA1, which include an E3 ubiquitin ligase activity at the N-terminus and a protein-protein interaction surface at the C-terminus, are critical to the function of this protein in the response to DNA damage.

The 60-kDa heat shock protein (HSP60), an endogenous ligand for the toll-like 4 receptor, is generated in response to inflammation, tissue injury, and/or stress and stimulates macrophages to produce cytotoxic and proinflammatory mediators including nitric oxide, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12. In the present studies we report that HSP60 is an effective inducer of cyclooxygenase-2 (COX-2) in macrophages, as well as endothelial cells. In both cell types, the synthesis of COX-2 was coordinate with induction of nitric oxide synthase (NOS)-2 and with nitric oxide production. With the use of promoter constructs in transient transfection assays, optimal expression of COX-2 in macrophages was found to require nuclear factor (NF)-kappaB, the cAMP-response element (CRE), and NF-IL-6, but not the E-box. Mobility shift assays revealed that HSP60 induced NF-kappaB and CRE binding activity, while CCAAT/enhancer binding protein (C/EBP), which binds to NF-IL-6, was constitutively active in the cells. Both c-Jun and CRE binding protein (CREB) bound to the CRE, while C/EBP-beta bound to NF-IL-6. These data indicate that NF-kappaB, C/EBP-beta, c-Jun, and CREB are important in HSP60-induced expression of COX-2. The c-Jun-NH(2)-terminal kinase (JNK), p44/42 mitogen-activated protein (MAP) kinase [extracellular signal-regulated kinase 1/2 (ERK1/2)], and p38 MAP kinase were rapidly activated by HSP60 in the macrophages. PD-98059, an inhibitor of phosphorylation of ERK1/2, caused a marked inhibition of HSP60-induced COX-2 and NOS-2 expression. Unexpectedly, SB-203580, a p38 kinase antagonist, was found to block HSP60-induced expression of COX-2, but not NOS-2. These data indicate that both ERK1/2 kinase and p38 kinase play a role in regulating HSP60-induced expression of COX-2.

Recent evidence suggests that the breast and ovarian cancer susceptibility gene product BRCA1 is involved in at least two fundamental cellular processes: transcriptional regulation and DNA repair. However, the mechanism of action of BRCA1 in either of these processes is still unknown. Here, we report the characterization of a disease-predisposing allele of BRCA1, identified in a family with several cases of ovarian cancer, coding for a protein that displays temperature-sensitive activity in transcriptional activation. The mutant protein differs from the wild type protein at a single amino acid, R1699W that occurs in a region at the N-terminal BRCT domain that is highly conserved among BRCA1 homologs. When the C-terminus of the mutant protein (aa 1560-1863) was fused to a heterologous GAL4 DNA-binding domain and expressed in yeast or mammalian cells, it was able to activate transcription of a reporter gene to levels observed for wild type BRCA1 at the permissive temperature (30 degrees C) but exhibited significantly less transcription activity at the restrictive temperature (37 degrees C or 39 degrees C). Our results indicate that the transcriptional activity of the R1699W mutant can be modulated as a function of temperature and provide a novel experimental approach which can be utilized to dissect the molecular mechanism(s) of BRCA1 in processes related to transcription.

BRCA1 is a tumor suppressor gene and germ line mutations account for the majority of familial cases of breast and ovarian cancer. There is mounting evidence that BRCA1 functions in DNA repair and transcriptional regulation. A major hurdle to dissect the role of BRCA1 is the lack of molecular reagents to carry out biochemical and genetic experiments. Therefore, we used random mutagenesis of the C-terminus of BRCA1 (aa 1560-1863) to generate temperature-sensitive (TS) mutants in transcription activation. We obtained 11 TS mutants in transcription that localized primarily to the hydrophobic core of the BRCT-N domain of BRCA1. One of the mutants, H1686Q, also displayed temperature-dependent transcription activation in human cells. These conditional mutants represent valuable tools to assess the role of BRCA1 in transcription activation.

Nitric oxide (NO) fluxes released from the surface of individual activated macrophages or cells localized in small aggregates were measured with a novel polarographic self-referencing microsensor. NO fluxes could be detected at distances from the cells of 100-500 microm. The initial flux and the distance from the cells at which NO could be detected were directly related to the number of cells in the immediate vicinity of the probe releasing NO. Thus, whereas NO fluxes of approximately 1 pmol. cm(-2). s(-1) were measured from individual macrophages, aggregates composed of groups of cells varying in number from 18 to 48 cells produced NO fluxes of between approximately 4 and 10 pmol. cm(-2). s(-1). NO fluxes required the presence of L-arginine. Signals were significantly reduced by the addition of hemoglobin and by N-nitro-L-arginine methyl ester. NO fluxes were greatest when the sensor was placed immediately adjacent to cell membranes and declined as the distance from the cell increased. The NO signal was markedly reduced in the presence of the protein albumin but not by either oxidized or reduced glutathione. A reduction in the NO signal was also noted after the addition of lipid micelles to the culture medium. These results demonstrate that NO can be detected at significant distances from the cell of origin. In addition, both proteins and lipids strongly influence the net movement of free NO from macrophages. This suggests that these tissue components play an important role in regulating the biological activity of NO.

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.