Phthalates, widely used in flexible plastics and consumer products, have become ubiquitous contaminants worldwide. This study evaluated the acute toxicity and estrogenic endocrine disrupting activity of butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), bis(2-ethylhexyl) phthalate (DEHP), diisodecyl phthalate (DIDP), diisononyl phthalate (DINP), di-n-octyl phthalate (DNOP) and their mixtures. Using a 72 h zebrafish embryo toxicity test, the LC50 values of BBP, DBP and a mixture of the six phthalates were found to be 0.72, 0.63 and 0.50 ppm, respectively. The other four phthalates did not cause more than 50% exposed embryo mortality even at their highest soluble concentrations. The typical toxicity symptoms caused by phthalates were death, tail curvature, necrosis, cardio edema and no touch response. Using an estrogen-responsive ChgH-EGFP transgenic medaka (Oryzias melastigma) eleutheroembryos based 24 h test, BBP demonstrated estrogenic activity, DBP, DEHP, DINP and the mixture of the six phthalates exhibited enhanced-estrogenic activity and DIDP and DNOP showed no enhanced- or anti-estrogenic activity. These findings highlighted the developmental toxicity of BBP and DBP, and the estrogenic endocrine disrupting activity of BBP, DBP, DEHP and DINP on intact organisms, indicating that the widespread use of these phthalates may cause potential health risks to human beings.

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We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)(2)(phen-ITC)](PF(6)) (Ir-ITC; Hpq=2-phenylquinoline, phen-ITC=5-isothiocyanate-1,10-phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir-ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir-ITC with live-cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage-dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir-ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.

Hypoxia is an important environmental stressor leading to endocrine disruption and reproductive impairment in fish. Although the hypoxia-inducible factor 1 (HIF-1) is known to regulate the transcription of various genes mediating oxygen homeostasis, its role in modulating steroidogenesis-related gene expression remains poorly understood. In this study, the regulatory effect of HIF-1 on the expression of 9 steroidogenic enzyme genes was investigated in zebrafish embryos using a “gain-of-function and loss-of-function” approach. Eight of the genes, CYP11a, CYP11b2, 3β-HSD, HMGCR, CYP17a1, 17β-HSD2, CYP19a, and CYP19b, were found to be differentially upregulated at 24 and 48 hpf following zHIF-1α-ΔODD overexpression (a mutant zebrafish HIF-1α protein with proline-414 and proline-557 deleted). Knockdown of zHIF-1α also affected the expression pattern of the steroidogenic enzyme genes. Overexpression of zHIF-1α and hypoxia exposure resulted in downregulated StAR expression but upregulated CYP11a and 3β-HSD expression in zebrafish embryos. Conversely, the expression patterns of these 3 genes were reversed in embryos in which zHIF-1α was knocked down under normoxia, suggesting that these 3 genes are regulated by HIF-1. Overall, the findings from this study indicate that HIF-1–mediated mechanisms are likely involved in the regulation of specific steroidogenic genes.