Repeated dose toxicity study to assess the safety and reproductive/developmental safety of 7-methyl-2H-1,5-Benzodioxepin-3(4H)-One (Calone®) and the read across approach to its biodegradation metabolite 7-methyl-2H-1,5-benzodioxepin-3-ol (Calol).

7-Methyl-2H-1,5-benzodioxepin-3(4H)-one (Calone®) is used in fragrances to impart a marine note. It is produced industrially at volumes requiring repeated dose and developmental/reproductive toxicology data (OECD TG 422) under European chemicals legislation (i.e., REACH). Additionally, Japanese chemicals legislation requires evaluation of Calone® biodegradability and identification of metabolites in an environmental biodegradation test. 7-Methyl-2H-1,5-benzodioxepin-3-ol (Calol) was the sole metabolite identified following biodegradation and a 28-day repeated dose toxicity study (OECD TG 407) would normally be required to support registration in Japan. The current paper presents results showing no adverse effects in the parental, reproductive, or developmental phases of an OECD TG 422 study following dietary administration of Calone® to rats at targeted doses of up to 1000 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was the highest administered dose of 791 and 922 mg/kg/day for males and females, respectively. An in vitro metabolism study conducted with rat and human liver microsomes demonstrated that greater than 90% of Calone® was metabolically reduced into Calol, the same metabolite observed in the environmental biodegradation test. Accordingly, the results from the OECD TG 422 study with Calone® are directly applicable to Calol and it would be expected to have the same NOAEL.

Cellular Stress and p53-Associated Apoptosis by Juniperus communis L. Berry Extract Treatment in the Human SH-SY5Y Neuroblastoma Cells.

Plant phenolics have shown to activate apoptotic cell death in different tumourigenic cell lines. In this study, we evaluated the effects of juniper berry extract (Juniperus communis L.) on p53 protein, gene expression and DNA fragmentation in human neuroblastoma SH-SY5Y cells. In addition, we analyzed the phenolic composition of the extract. We found that juniper berry extract activated cellular relocalization of p53 and DNA fragmentation-dependent cell death. Differentially expressed genes between treated and non-treated cells were evaluated with the cDNA-RDA (representational difference analysis) method at the early time point of apoptotic process when p53 started to be activated and no caspase activity was detected. Twenty one overexpressed genes related to cellular stress, protein synthesis, cell survival and death were detected. Interestingly, they included endoplasmic reticulum (ER) stress inducer and sensor HSPA5 and other ER stress-related genes CALM2 and YKT6 indicating that ER stress response was involved in juniper berry extract mediated cell death. In composition analysis, we identified and quantified low concentrations of fifteen phenolic compounds. The main groups of them were flavones, flavonols, phenolic acids, flavanol and biflavonoid including glycosides of quercetin, apigenin, isoscutellarein and hypolaetin. It is suggested that juniper berry extract induced the p53-associated apoptosis through the potentiation and synergism by several phenolic compounds.

Minocycline protects SH-SY5Y cells from 6-hydroxydopamine by inhibiting both caspase-dependent and -independent programmed cell death.

Minocycline, a tetracyclic antibiotic, exerts both antiinflammation by acting on microglia and a direct protection on neurons by inhibiting the apoptotic machinery at various levels. However, we are not aware of any study investigating the effects of minocycline on caspase-independent programmed cell death (PCD) pathways. This study investigated these alternative pathways in SH-SY5Y cells, a human dopaminergic cell line, challenged with 6-hydroxydopamine (6-OHDA). Minocycline exhibited neuroprotection and inhibition of the toxin-induced caspase-3-like activity, DNA fragmentation, and chromatin condensation, hallmarks of apoptosis. Moreover, we revealed that 6-OHDA also activated caspase-independent PCDs (such as paraptosis), which required de novo protein synthesis. Additionally, by separately monitoring caspase-dependent and caspase-independent pathways, we showed that inhibition of apoptosis only partially explained the protective effect of minocycline. Moreover, we observed that minocycline reduced the protein content of cells but, unexpectedly, increased the protein synthesis. These findings suggest that minocycline may actually increase protein degradation, so it may also accelerate the clearance of aberrant proteins. In conclusion, we report for the first time evidence indicating that minocycline may inhibit PCD pathways that are additional to conventional apoptosis.

Antioxidant, pro-oxidant and cytotoxic properties of parsley.

Parsley (Petroselinum crispum) leaves were macerated with a mixture of methanol: water: acetic acid to produce a crude extract which was then defatted with (40°-60°) petrol. Antioxidant activity of the extract was evaluated using a battery of in vitro assays, viz., iron(iii) reduction, iron(ii) chelation and free radical scavenging assays. Evaluation of the pro-oxidant activity of the extract was based upon its effects upon DNA fragmentation and protein carbonylation. Cytotoxicity and apoptotic effects of the extract were determined in non-cancerous CV1-P fibroblast and cancerous A375 melanoma cells using MTT and LDH tests and caspase 3-like activity assay. The highest concentration, 2.0 mg ml(-1), decreased the viability of both cell lines, however, the cancerous melanoma cells were slightly susceptible to the effects. The observed cytotoxicity was not due to the caspase 3 activity. In conclusion, the toxicity might be explained by the pro-oxidative activity of components within the extract against proteins and/or DNA but it is not related to caspase 3-dependent apoptosis within cells.