Characterization of the Glutathione S-Transferases Involved in Styrene Degradation in Gordonia rubripertincta CWB2.

The glutathione S-transferases carried on the plasmid for the styrene-specific degradation pathway in the Actinobacterium Gordonia rubripertincta CWB2 were heterologously expressed in Escherichia coli. Both enzymes were purified via affinity chromatography and subjected to activity investigations. StyI and StyJ displayed activity toward the commonly used glutathione S-transferase model substrate 1-chloro-2,4-dinitrobenzene (CDNB) with Km values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and Vmax values of 0.0158 ± 0.0002 and 0.348 ± 0.008 U mg-1 for StyI and StyJ, respectively. The conversion of the natural substrate styrene oxide to the intermediate (1-phenyl-2-hydroxyethyl)glutathione was detected for StyI with 48.3 ± 2.9 U mg-1. This elucidates one more step in the not yet fully resolved styrene-specific degradation pathway of Gordonia rubripertincta CWB2. A characterization of both purified enzymes adds more insight into the scarce research field of actinobacterial glutathione S-transferases. Moreover, a sequence and phylogenetic analysis puts both enzymes into a physiological and evolutionary context. IMPORTANCE Styrene is a toxic compound that is used at a large scale by industry for plastic production. Bacterial degradation of styrene is a possibility for bioremediation and pollution prevention. Intermediates of styrene derivatives degraded in the styrene-specific pathways are precursors for valuable chemical compounds. The pathway in Gordonia rubripertincta CWB2 has proven to accept a broader substrate range than other bacterial styrene degraders. The enzymes characterized in this study, distinguish CWB2s pathway from other known styrene degradation routes and thus might be the main key for its ability to produce ibuprofen from the respective styrene derivative. A biotechnological utilization of this cascade could lead to efficient and sustainable production of drugs, flavors, and fragrances. Moreover, research on glutathione metabolism in Actinobacteria is rare. Here, a characterization of two glutathione S-transferases of actinobacterial origin is presented, and the utilization of glutathione in the metabolism of an Actinobacterium is proven.

Molecular Recognition and Imaging of Human Telomeric G-Quadruplex DNA in Live Cells: A Systematic Advancement of Thiazole Orange Scaffold To Enhance Binding Specificity and Inhibition of Gene Expression.

A series of fluorescent ligands, which were systematically constructed from thiazole orange scaffold, was investigated for their interactions with G-quadruplex structures and antitumor activity. Among the ligands, compound 3 was identified to exhibit excellent specificity toward telomere G4-DNA over other nucleic acids. The affinity of 3-Htg24 was almost 5 times higher than that of double-stranded DNA and promoter G4-DNA. Interaction studies showed that 3 may bind to both G-tetrad and the lateral loop near the 5'-end. The intracellular colocalization with BG4 and competition studies with BRACO19 reveal that 3 may interact with G4-structures. Moreover, 3 reduces the telomere length and downregulates hTERC and hTERT mRNA expression in HeLa cells. The cytotoxicity of 3 against cancer cells (IC50 = 12.7-16.2 µM) was found to be generally higher than noncancer cells (IC50 = 52.3 µM). The findings may support that the ligand is telomere G4-DNA specific and may provide meaningful insights for anticancer drug design.

Dehydrozingerone, a Curcumin Analog, as a Potential Anti-Prostate Cancer Inhibitor In Vitro and In Vivo.

Curcumin (Cur) exhibits biological activities that support its candidacy for cancer treatment. However, there are limitations to its pharmacological effects, such as poor solubility and bioavailability. Notably, the use of Cur analogs has potential for addressing these limitations. Dehydrozingerone (DZG) is a representative of the half-chemical structure of Cur, and many reports have indicated that it is anticancer in vitro. We, therefore, have hypothesized that DZG could inhibit prostate cancer progression both in vitro and in vivo. Results revealed that DZG decreased cell proliferation of rat castration-resistant prostate cancer, PLS10 cells, via induction of the cell cycle arrest in the G1 phase in vitro. In the PLS10 xenograft model, DZG significantly decreased the growth of subcutaneous tumors when compared to the control via the inhibition of cell proliferation and angiogenesis. To prove that DZG could improve the limitations of Cur, an in vivo pharmacokinetic was determined. DZG was detected in the serum at higher concentrations and remained up to 3 h after intraperitoneal injections, which was longer than Cur. DZG also showed superior in vivo tissue distribution than Cur. The results suggest that DZG could be a candidate of the Cur analog that can potentially exert anticancer capabilities in vivo and thereby improve its bioavailability.

Quaternized styryl-azinium fluorophores as cellular RNA-binders.

The capability of three quaternized styryl-azinium iodides to bind cellular RNA has been tested by means of Fluorescence Confocal Microscopy imaging of stained MCF-7 cells treated with RNase. Their association constants have been estimated through spectrophotometric and fluorimetric titrations with tRNA and compared to their affinity toward DNA. Transient absorption spectroscopy with femtosecond resolution confirmed the binding of the investigated compounds with tRNA and shed new light on the excited state dynamics of their complexes, by revealing a significant lengthening of the lifetime of S1 upon complexation, which parallels the fluorescence quantum yield enhancement.

Stereoselective Activity of 1-Propargyl-4-styrylpiperidine-like Analogues That Can Discriminate between Monoamine Oxidase Isoforms A and B.

The resurgence of interest in monoamine oxidases (MAOs) has been fueled by recent correlations of this enzymatic activity with cardiovascular, neurological, and oncological disorders. This has promoted increased research into selective MAO-A and MAO-B inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be achieved by geometric isomers of cis- and trans-1-propargyl-4-styrylpiperidines. While the cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only the MAO-B isoform. The inhibition was studied by kinetic analysis, UV-vis spectrum measurements, and X-ray crystallography. The selective inhibition of the MAO-A and MAO-B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous system disorders. This study represents a unique case of stereoselective activity of cis/trans isomers that can discriminate between structurally related enzyme isoforms.

In vitro antitumor activity, ADME-Tox and 3D-QSAR of synthesized and selected natural styryl lactones.

Prostate cancer is a common cause of death in men and a novel treating methods should be developed. In order to find a new drug for prostate cancer, a series of novel conformationally constrained analogues of (+)-goniofufurone and 7-epi-(+)-goniofufurone, as well as the newly synthesized styryl lactones containing the cinnamic acid ester groups were evaluated for in vitro cytotoxicity against prostate cancer cell (PC-3). Furthermore, prediction of physicochemical characteristics and drugability as well as in silico ADME-Tox tests of investigated compounds were performed. The 3D-QSAR model was established using the comparative molecular field analysis method. According to obtained results, the tricyclic compounds 9 and 10 had the highest potency with IC50 < 20 µM. Evaluation of structural features through 3D-QSAR model identified steric field feature on the cinnamic acid ester groups at C-7 as a crucial for the cytotoxic activity. This research suggests that most of the analysed compounds have desirable properties for drug candidates and high potential in drug development, which recommend them for further research in treatment of prostate cancer. Furthermore, obtained 3D-QSAR model is able to successfully identify styryl lactones that have significant cytotoxic activity and provide information for screening and design of novel inhibitors against PC-3 cell line that could be used as drugs in treatment of the prostate cancer.

Covalent Attachment of Fibronectin onto Emulsion-Templated Porous Polymer Scaffolds Enhances Human Endometrial Stromal Cell Adhesion, Infiltration, and Function.

A novel strategy for the surface functionalization of emulsion-templated highly porous (polyHIPE) materials as well as its application to in vitro 3D cell culture is presented. A heterobifunctional linker that consists of an amine-reactive N-hydroxysuccinimide ester and a photoactivatable nitrophenyl azide, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), is utilized to functionalize polyHIPE surfaces. The ability to conjugate a range of compounds (6-aminofluorescein, heptafluorobutylamine, poly(ethylene glycol) bis-amine, and fibronectin) to the polyHIPE surface is demonstrated using fluorescence imaging, FTIR spectroscopy, and X-ray photoelectron spectroscopy. Compared to other existing surface functionalization methods for polyHIPE materials, this approach is facile, efficient, versatile, and benign. It can also be used to attach biomolecules to polyHIPE surfaces including cell adhesion-promoting extracellular matrix proteins. Cell culture experiments demonstrated that the fibronectin-conjugated polyHIPE scaffolds improve the adhesion and function of primary human endometrial stromal cells. It is believed that this approach can be employed to produce the next generation of polyHIPE scaffolds with tailored surface functionality, enhancing their application in 3D cell culture and tissue engineering whilst broadening the scope of applications to a wider range of cell types.

Orientation of a Diagnostic Ligand Bound to Macroscopically Aligned Amyloid-ß Fibrils Determined by Solid-State NMR.

With amyloid diseases poised to become a major health burden in countries with aging populations, diagnostic molecules that aid the detection of amyloid in vitro and in vivo are of considerable clinical value. Understanding how such ligands recognize their amyloid targets would help to design diagnostics that target specific amyloid types associated with a particular disease, but methods to provide comprehensive information are underdeveloped. Here, solid-state NMR is used to determine the molecular orientation of the amyloid diagnostic 1-fluoro-2,5-bis[( E)-3-carboxy-4-hydroxystyryl]-benzene (FSB) when bound to fibrils of the Alzheimer's amyloid-ß polypeptide aligned on a planar substrate. The 19F NMR spectrum of the aligned complex reveals that FSB is oriented approximately parallel with the fibril long axis and bridges four hydrogen-bonded ß-sheets. In addition to providing atomic details to aid the design of amyloid-specific diagnostics, this approach will also illuminate the molecular mechanisms of accessory molecules in amyloid disease.

Assessing molecular initiating events (MIEs), key events (KEs) and modulating factors (MFs) for styrene responses in mouse lungs using whole genome gene expression profiling following 1-day and multi-week exposures.

Styrene increased lung tumors in mice at chronic inhalation exposures of 20ppm and greater. MIEs, KEs and MFs were examined using gene expression in three strains of male mice (the parental C57BL/6 strain, a CYP2F2(-/-) knock out and a CYP2F2(-/-) transgenic containing human CYP2F1, 2A13 and 2B6). Exposures were for 1-day and 1, 4 and 26weeks. After 1-day exposures at 1, 5, 10, 20, 40 and 120ppm significant increases in differentially expressed genes (DEGs) occurred only in parental strain lungs where there was already an increase in DEGs at 5ppm and then many thousands of DEGs by 120ppm. Enrichment for 1-day and 1-week exposures included cell cycle, mitotic M-M/G1 phases, DNA-synthesis and metabolism of lipids and lipoproteins pathways. The numbers of DEGs decreased steadily over time with no DEGs meeting both statistical significance and fold-change criteria at 26weeks. At 4 and 26weeks, some key transcription factors (TFs) - Nr1d1, Nr1d2, Dbp, Tef, Hlf, Per3, Per2 and Bhlhe40 - were upregulated (|FC|>1.5), while others - Npas, Arntl, Nfil3, Nr4a1, Nr4a2, and Nr4a3 - were down-regulated. At all times, consistent changes in gene expression only occurred in the parental strain. Our results support a MIE for styrene of direct mitogenicity from mouse-specific CYP2F2-mediated metabolites activating Nr4a signaling. Longer-term MFs include down-regulation of Nr4a genes and shifts in both circadian clock TFs and other TFs, linking circadian clock to cellular metabolism. We found no gene expression changes indicative of cytotoxicity or activation of p53-mediated DNA-damage pathways.

RXRα ligand Z-10 induces PML-RARα cleavage and APL cell apoptosis through disrupting PML-RARα/RXRα complex in a cAMP-independent manner.

The major oncogenic driver of acute promyelocytic leukemia (APL) is the fusion protein PML-RARα originated from the chromosomal translocation t(15;17). All-trans retinoic acid (ATRA) and arsenic trioxide cure most patients by directly targeting PML-RARα. However, major issues including the resistance of ATRA and arsenic therapy still remain in APL clinical management. Here we showed that compound Z-10, a nitro-ligand of retinoid X receptor α (RXRα), strongly promoted the cAMP-independent apoptosis of both ATRA- sensitive and resistant NB4 cells via the induction of caspase-mediated PML-RARα degradation. RXRα was vital for the stability of both PML-RARα and RARα likely through the interactions. The binding of Z-10 to RXRα dramatically inhibited the interaction of RXRα with PML-RARα but not with RARα, leading to Z-10's selective induction of PML-RARα but not RARα degradation. Z-36 and Z-38, two derivatives of Z-10, had improved potency of inducing PML-RARα reduction and NB4 cell apoptosis. Hence, RXRα ligand Z-10 and its derivatives could target both ATRA- sensitive and resistant APL cells through their distinct acting mechanism, and are potential drug leads for APL treatment.

(68)Ga-Bivalent Polypegylated Styrylpyridine Conjugates for Imaging Aß Plaques in Cerebral Amyloid Angiopathy.

Aß plaques deposited on blood vessels are associated with cerebral amyloid angiopathy (CAA). In an effort to selectively map these Aß plaques, we are reporting a new series of (68)Ga labeled styrylpyridine derivatives with high molecular weights. In vitro binding to Aß plaques in post-mortem Alzheimer's disease (AD) brain tissue showed that these (68)Ga labeled bivalent styrylpyridines displayed good affinities and specificity (Ki < 30 nM). In vitro autoradiography using post-mortem AD brain sections showed specific binding of these (68)Ga complexes to Aß plaques. Biodistribution studies in normal mice showed very low initial brain uptakes (<0.3% dose/g) indicating a low blood-brain barrier (BBB) penetration. The preliminary results suggest that (68)Ga labeled bivalent styrylpyridines may be promising candidates as PET imaging radiotracers for detecting CAA.

Reversible S-nitrosylation limits over synthesis of fungal styrylpyrone upon nitric oxide burst.

Nitric oxide (NO) is known to be involved in modulating production of styrylpyrone polyphenols in the basidiomycete Inonotus obliquus. However, it remains unknown how NO orchestrates fungal styrylpyrone biosynthesis. Here, we show that a transient NO burst correlated with an enhanced expression of phenylalanine ammonia lyase (PAL), 4-coumarate CoA ligase (4CL), and styrylpyrone synthase (SPS), the key enzymes involved in styrylpyrone biosynthesis, and subsequently an increased production of styrylpyrone polyphenols. In parallel, the NO burst also resulted in S-nitrosylation of PAL, 4CL, and SPS, which compromised their enzymatic activities mediating a post-translational feedback mechanism that keeps NO-dependent transcriptional activation in check. Moreover, dysfunction of thioredoxin reductase (TrxR) further increased the formation of S-nitrosylated proteins, implicating the significance of the Trx system in maintaining a low level of protein-nitrosothiols. Three thioredoxin-like proteins (TrxLs) from I. obliquus show in vitro denitrosylation potential toward S-nitrosylated proteins via trans-denitrosylation or mixed disulfide intermediates. Thus, S-nitrosylation triggered by the NO burst limits over production of fungal styrylpyrone polyphenols, and denitrosylation by TrxLs that act in concert with TrxR play a key role in maintaining redox balance and orchestrating catalytic activities of the enzymes engaged in styrylpyrone synthetic metabolism.

Fluorescence-Based Automated Screening Assay for the Study of the pH-Sensitive Channel ASIC1a.

Acid-sensing ion channel 1a (ASIC1a) is involved in several pathologies, including neurodegenerative and neuroinflammatory disorders, stroke, epilepsy, and inflammatory pain. ASIC1a has been the subject of intense drug discovery programs devoted to the development of new pharmacological tools for its modulation. However, these efforts to generate new compounds have faced the lack of an efficient screening procedure. In the past decades, improvements in screening technologies and fluorescent sensors for the study of ion channels have provided new opportunities in this field. Unfortunately, ASIC1a is mainly a Na(+) permeable channel and undergoes desensitization after its activation, two features that make the use of the available screening procedures problematic. We propose here a novel screening approach for the study of ASIC1a activity in full automation. Our method is based on the stimulation of ASIC1a-expressing cells by protons and the use of electrochromic fluorescent voltage sensors as a readout of ion channel activation. This method will prove to be useful for drug screening programs aimed at ASIC1a modulation.

Novel PI3K/AKT targeting anti-angiogenic activities of 4-vinylphenol, a new therapeutic potential of a well-known styrene metabolite.

The pneumo- and hepato-toxicity of 4-vinylphenol (4VP), a styrene metabolite, has been previously reported. Nevertheless, the present study reported the novel anti-angiogenic activities of 4VP which was firstly isolated from the aqueous extract of a Chinese medicinal herb Hedyotis diffusa. Our results showed that 4VP at non-toxic dose effectively suppressed migration, tube formation, adhesion to extracellular matrix proteins, as well as protein and mRNA expressions of metalloproteinase-2 of human endothelial cells (HUVEC and HMEC-1). Investigation of the signal transduction revealed that 4VP down-regulated PI3K/AKT and p38 MAPK. Besides, 4VP interfered with the phosphorylation of ERK1/2, the translocation and expression of NFkappaB. In zebrafish embryo model, the new blood vessel growth was significantly blocked by 4VP (6.25-12.5 µg/mL medium). The VEGF-induced blood vessel formation in Matrigel plugs in C57BL/6 mice was suppressed by 4VP (20-100 µg/mL matrigel). In addition, the blood vessel number and tumor size were reduced by intraperitoneal 4VP (0.2-2 mg/kg) in 4T1 breast tumor-bearing BALB/c mice, with doxorubicin as positive control. Together, the in vitro and in vivo anti-angiogenic activities of 4VP were demonstrated for the first time. These findings suggest that 4VP has great potential to be further developed as an anti-angiogenic agent.

Health impact assessment of decreases in PM10 and ozone concentrations in the Mexico City Metropolitan Area: A basis for a new air quality management program / Evaluación de impacto en salud ante reducciones de PM10 y ozono en la Zona Metropolitana del Valle de México: Base para un nuevo programa de calidad del aire

Objective. To conduct a health impact assessment (HIA) to quantify health benefits for several PM and O3 air pollution reduction scenarios in the Mexico City Metropolitan Area (MCMA). Results from this HIA will contribute to the scientific support of the MCMA air quality management plan (PROAIRE) for the period 2011-2020. Materials and methods. The HIA methodology consisted of four steps: 1) selection of the air pollution reduction scenarios, 2) identification of the at-risk population and health outcomes for the 2005 baseline scenario, 3) selection of concentration-response functions and 4) estimation of health impacts. Results. Reductions of PM10 levels to 20 μg/m³ and O3 levels to 0.050ppm (98 µg/m³) would prevent 2300 and 400 annual deaths respectively. The greatest health impact was seen in the over-65 age group and in mortality due to cardiopulmonary and cardiovascular disease. Conclusion. Improved air quality in the MCMA could provide significant health benefits through focusing interventions by exposure zones.

Objetivo. Realizar una evaluación de impacto en salud (EIS) que documente los beneficios en salud ante diversos escenarios de reducción de PM10 y O3 en el aire de la Zona Metropolitana del Valle de México (ZMVM). Los resultados contribuyen al sustento científico del plan de gestión de calidad del aire (PROAIRE 2011-2020). Material y métodos. La metodología de EIS comprende cuatro pasos: 1) selección de los escenarios de reducción, 2) identificación de la población en riesgo y de los eventos en salud para el año basal 2005, 3) selección de las funciones de concentración-respuesta y 4) estimación del impacto en la salud. Resultados. Reducciones de PM10 a 20μg/m³ y de O3 a 0.050ppm (98 µg/m³) evitarían, respectivamente, cerca de 2 300 y 400 muertes por año. El mayor impacto se observa en el grupo de más de 65 años y en la mortalidad por causas cardiopulmonares y cardiovasculares. Conclusiones. Mejorar la calidad del aire en la ZMVM podría reflejar importantes beneficios para la salud focalizados por zonas o áreas de exposición.

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties.

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4-6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H2O2, MPP+ and MnCl2 were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.

Determination of the glucuronide metabolite of ON 013100, a benzylstyrylsulfone antineoplastic drug, in colon cancer cells using LC/MS/MS.

ON 013100, (E)-2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzyl sulfone, is a potent kinase inhibitor whose phosphate form is in Phase I clinical trials in lymphoma and acute lymphoid leukemia. The objectives were to: (a) investigate the possible presence of the glucuronide metabolite of the drug in two representative colon cancer cell lines, a drug resistant (colo-205) and a drug sensitive (colo-320); (b) quantify the glucuronide metabolite and the unchanged drug in the cells after treatment with ON 013100. The glucuronide was synthesized and a selective LC/MS/MS method was developed and validated for the characterization and quantification of the metabolite. The glucuronide metabolite (570.6 Da) was found in the drug-resistant cells upon a 1h incubation with ON 013100 (20 µg/ml). After treatment with the drug, the concentration of the metabolite gradually decreased from 0.84 µg/ml at 0 h through 0.21 µg/ml at 6h to below detection limit of 8.0 ng/ml at 9 h. No glucuronide metabolite was detected in the drug-sensitive cells. The concentrations of intact ON 013100 in the drug-resistant cells gradually decreased from 0.41 µg/ml (0 h) to 0.06 µg/ml (9 h). The corresponding concentrations of the intact drug in the drug-sensitive cells were from 2.88 µg/ml to 0.94 µg/ml.

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.

Disposition and metabolism of cumene in F344 rats and B6C3F1 mice.

Cumene is a high-production volume chemical that has been shown to be a central nervous system depressant and has been implicated as a long-term exposure carcinogen in experimental animals. The absorption, distribution, metabolism, and excretion of [(14)C]cumene (isopropylbenzene) was studied in male rats and mice of both sexes after oral or intravenous administration. In both species and sexes, urine accounted for the majority of the excretion (typically ≥ 70%) by oral and intravenous administration. Enterohepatic circulation of cumene and/or its metabolites was indicated because 37% of the total dose was excreted in bile in bile duct-cannulated rats with little excreted in normal rats. The highest tissue (14)C levels in rats were observed in adipose tissue, liver, and kidney with no accumulation observed after repeat dosing up to 7 days. In contrast, mice contained the highest concentrations of (14)C at 24 h after dosing in the liver, kidney, and lung, with repeat dosing accumulation of (14)C observed in these tissues as well as in the blood, brain, heart, muscle, and spleen. The metabolites in the expired air, urine, bile, and microsomes were characterized with 16 metabolites identified. The volatile organics in the expired air comprised mainly cumene and up to 4% α-methylstyrene. The major urinary and biliary metabolite was 2-phenyl-2-propanol glucuronide, which corresponded with the main microsomal metabolite being 2-phenyl-2-propanol.