Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans.

Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.

Comparison of the Effects of Contractubex Gel and Benzothiazole After Topical Application in an Experimental Model of Epidural Fibrosis in Rats.

BACKGROUND: Postoperative epidural adhesion is a frequent cause of failed back surgery syndrome, manifesting with back and leg pain or neurologic deficits. Development of preventive measures for epidural adhesion after laminectomy is critical to improve outcomes of lumbar surgery. We hypothesized that positive effects of topical application of Contractubex (CTX) gel and benzothiazole (BT) individually and in combination could aid in preventing epidural fibrosis in a rat laminectomy model. METHODS: Rats were randomly assigned to 2 control and 5 experimental groups (n = 8 for each group). The control(-) group received no surgery, whereas the control(+) group underwent laminectomy without any drug administration. In experimental groups, study agents applied to dura mater after laminectomy were 100mgCTX, 2.5%BT, 5%BT; 100mgCTXplus2.5%BT, and 100mgCTXplus5%BT. Laminectomy was performed at the L3 level for all rats. The extent of epidural fibrosis was assessed 4 weeks later macroscopically and histopathologically. Hepatic and renal toxicity of study drugs was assessed histopathologically. RESULTS: Topical CTX and BT individually and in combination reduced epidural fibrosis after laminectomy in rats. Although a meaningful decrease of epidural fibrosis with individual application of CTX and BT (2.5% or 5%) was obtained (P < 0.05), the effect of their combination was more pronounced without meaningful hepatic and renal toxicity (P < 0.05). CONCLUSIONS: Combined use of topical CTX and BT could be a potential therapy for epidural fibrosis. Further research with this agents for the prevention of epidural fibrosis is warranted.

A Nonbactericidal Zinc-Complexing Ligand as a Biofilm Inhibitor: Structure-Guided Contrasting Effects on Staphylococcus aureus Biofilm.

Zinc-complexing ligands are prospective anti-biofilm agents because of the pivotal role of zinc in the formation of Staphylococcus aureus biofilm. Accordingly, the potential of a thiosemicarbazone (compound C1) and a benzothiazole-based ligand (compound C4) in the prevention of S. aureus biofilm formation was assessed. Compound C1 displayed a bimodal activity, hindering biofilm formation only at low concentrations and promoting biofilm growth at higher concentrations. In the case of C4, a dose-dependent inhibition of S. aureus biofilm growth was observed. Atomic force microscopy analysis suggested that at higher concentrations C1 formed globular aggregates, which perhaps formed a substratum that favored adhesion of cells and biofilm formation. In the case of C4, zinc supplementation experiments validated zinc complexation as a plausible mechanism of inhibition of S. aureus biofilm. Interestingly, C4 was nontoxic to cultured HeLa cells and thus has promise as a therapeutic anti-biofilm agent. The essential understanding of the structure-driven implications of zinc-complexing ligands acquired in this study might assist future screening regimes for identification of potent anti-biofilm agents.

Effects of the 5HT2C antagonist SB242084 on the pramipexole-induced potentiation of water contrafreeloading, a putative animal model of compulsive behavior.

RATIONALE: In rats, quinpirole, a dopaminergic D2/D3 receptor agonist, elicits both hyperdipsia and water "contrafreeloading" (CFL), a putative model of compulsivity. The role of D3 receptors in this effect remains unclear. Clomipramine (CIM) was found to contrast both hyperdipsia and CFL, but the role of serotonin in this effect requires further investigation. OBJECTIVES: We studied the effects of the preferential D3 agonist pramipexole (PPX) in both models. Furthermore, we tested the sensitivity of PPX-induced CFL to CIM and to the 5HT2c antagonist SB242084. METHODS: In experiment 1, drinking was measured at 2 and 5 h after eight daily injections of PPX (0 to 1.0 mg/kg intraperitoneally). In the CFL study, every other third lever press, the rat was reinforced by the delivery of water. On days 1-6, water was only available upon lever pressing. On days 7-15, choice between response-contingent and free access was provided. PPX doses as in the experiment 1 were given. In two further experiments, PPX (0.5 mg/kg) was administered alone or in combination with CIM (5 or 10 mg/kg) or SB242084 (0.3 or 1.0 mg/kg). RESULTS: PPX did not produce hyperdipsia but enhanced spontaneous CFL. SB242084 attenuated PPX-induced CFL more effectively than CIM, restoring the preference for free access to water. CONCLUSIONS: CFL, but not polydipsia, was induced by preferential D3 activation, an effect prevented by 5HT2c receptor blockade. Since PPX interferes with decision making and 5HT2c receptor supersensitivity is involved in the expression of compulsive behaviors, this study supports the compulsive nature of dopaminergic-induced CFL.

Pharmacophore-based drug design and biological evaluation of novel ABCB1 inhibitors.

Overexpression of ABCB1 is one of major barriers for multidrug resistance in chemotherapy and limits drug oral bioavailability. Inhibition of ABCB1 would sensitize multidrug resistance in clinical cancer chemotherapy. With this aim, a 3D pharmacophore model was created based on known ABCB1 inhibitors with correlation coefficient of 0.94, comprising three hydrophobic features and one hydrogen bond acceptor. It was further validated and used to search our in-house 3D database for potential ABCB1 inhibitors. The inhibitory activities of the best hits were evaluated by several biological assays, such as rhodamine 123 accumulation assay, chemosensitization assay, multidrug resistance 1-Madin-Darby canine kidney cells/Madin-Darby canine kidney cells permeability assay. Finally, compounds YZ-3 and YZ-16 were identified as potential leads to be developed in the designing of novel potent ABCB1 inhibitors.

Synthesis and biological activity of hydroxy substituted phenyl-benzo[d]thiazole analogues for antityrosinase activity in B16 cells.

In this study, we synthesized hydroxy and/or alkoxy substituted phenyl-benzo[d]thiazole derivatives using substituted benzaldehydes and 2-aminothiophenol in MeOH. The structures of these compounds were established by (1)H and (13)CNMR and mass spectral analyzes. All synthesized compounds were evaluated for their mushroom tyrosinase inhibition activity. Out the 12 generated compounds, 2a and 2d exhibited much higher tyrosinase inhibition activity (45.36-73.07% and 49.94-94.17% at 0.01-20 µM, respectively) than kojic acid (9.29-50.80% at 1.25-20 µM), a positive control. The cytotoxicity of 2a and 2d was evaluated using B16 cells and the compounds were found to be nontoxic. Compounds 2a and 2d were also demonstrated to be potent mushroom tyrosinase inhibitors, displaying IC(50) values of 1.14±0.48 and 0.01±0.0002 µM, respectively, compared with kojic acid, which has an IC(50) value of 18.45±0.17 µM. We also predicted the tertiary structure of tyrosinase, simulated the docking with compounds 2a and 2d and confirmed that the compounds strongly interact with mushroom tyrosinase residues. Kinetic plots showed that 2a and 2d are competitive tyrosinase inhibitors. Substitutions with a hydroxy group at R(3) or both R(3) and R(1) of the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase. We further found that compounds 2a and 2d inhibit melanin production and tyrosinase activity in B16 cells. These results may assist in the development of new potent tyrosinase inhibitors against hyperpigmentation.

Synthesis and anticonvulsant activity of 7-alkoxy-triazolo-[3, 4-b]benzo[d]thiazoles.

The present study describes the chemical synthesis and anticonvulsant activity evaluation of a series of 7-alkoxy-triazolo-[3, 4-b]benzo[d]thiazoles. Most compounds exhibited good anticonvulsant activity in the Maximal electroshock (MES) test. And the structure-activity relationships (SAR) were analyzed. Among the compounds studied, 7-octyloxy-triazolo-[3, 4-b]benzo[d]thiazole (5g) was found to be the most potent compound with a median effective dose (ED(50)) value of 8.0 mg/kg and a protective index (PI) value of 15.0, possessing better anticonvulsant activity and higher safety than marketed drugs carbamazepine and phenytoin. The mechanism study of compound 5g showed that it displayed broad spectrum activity in several models, and it is likely to have several mechanisms of action (including inhibiting voltage-gated ion channels and GABAergic activity).

Screening study for repeated dose and reproductive/developmental toxicity of rubber accelerator, N,N-dicyclohexyl-2-benzothiazolesulfenamide, in rats.

A screening study for a vulcanization accelerator N,N-dicyclohexyl-2-benzothiazole-sulfenamide (DCBS) was performed in rats. Rats were given DCBS by gavage daily at 0, 6, 25, 100, or 400 mg/kg. Males were dosed for a total of 44 days beginning 14 days before mating. Females were dosed for a total of 40-51 days beginning 14 days before mating to day 3 of lactation. Toxicologic changes were significantly noted only at 400 mg/kg. Three females died. An increased incidence of females showing decreased locomotor activity, soil of the lower abdominal fur, and reddish tears was observed. A lowered body weight was found in males and females. Increased urinary ketones and serum inorganic phosphorus and decreased serum glutamate pyruvate transaminase in males were found. Increased absolute and relative weights of the kidneys in males and decreased absolute weight of the thymus in both sexes were noted. Significant fatty degeneration of the renal tubular epithelia, vacuolation of the adrenocortical cells, and atrophy of the spleen were observed in females. Significant decreases in the gestation index, numbers of corpura lutea, implantations, pups born and pups born alive, live birth index, and viability index were detected. It is concluded that the No Observed Adverse Effect Levels (NOAELs) for repeat dose and reproductive/developmental toxicity are 100 mg kg-1 day-1 in this screening study.