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Jeffrey D. Laskin, Ph.D.


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Selective Targeting of Heme Protein in Cytochrome P450 and Nitric Oxide Synthase by Diphenyleneiodonium.

Mon, 02/19/2018 - 03:00

Related Articles Selective Targeting of Heme Protein in Cytochrome P450 and Nitric Oxide Synthase by Diphenyleneiodonium. Toxicol Sci. 2016 May;151(1):150-9 Authors: Szilagyi JT, Mishin V, Heck DE, Jan YH, Aleksunes LM, Richardson JR, Heindel ND, Laskin DL, Laskin JD Abstract Cytochrome P450 (CYP) enzymes mediate mixed-function oxidation reactions important in drug metabolism. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), binds flavin in cytochrome P450 reductase and inhibits CYP-mediated activity. DPI also inhibits CYP by directly interacting with heme. Herein, we report that DPI effectively inhibits a number of CYP-related monooxygenase reactions including NADPH oxidase, a microsomal enzyme activity that generates hydrogen peroxide in the absence of metabolizing substrates. Inhibition of monooxygenase by DPI was time and concentration dependent with IC50's ranging from 0.06 to 1.9 μM. Higher (4.6-23.9 μM), but not lower (0.06-1.9 μM), concentrations of DPI inhibited electron flow via cytochrome P450 reductase, as measured by its ability to reduce cytochrome c and mediate quinone redox cycling. Similar results were observed with inducible nitric oxide synthase (iNOS), an enzyme containing a C-terminal reductase domain homologous to cytochrome P450 reductase that mediates reduction of cytochrome c, and an N-terminal heme-thiolate oxygenase domain mediating nitric oxide production. Significantly greater concentrations of DPI were required to inhibit cytochrome c reduction by iNOS (IC50 = 3.5 µM) than nitric oxide production (IC50 = 0.16 µM). Difference spectra of liver microsomes, recombinant CYPs, and iNOS demonstrated that DPI altered heme-carbon monoxide interactions. In the presence of NADPH, DPI treatment of microsomes and iNOS yielded a type II spectral shift. These data indicate that DPI interacts with both flavin and heme in CYPs and iNOS. Increased sensitivity for inhibition of CYP-mediated metabolism and nitric oxide production by iNOS indicates that DPI targets heme moieties within the enzymes. PMID: 26880746 [PubMed - indexed for MEDLINE]

Categories: Publications from UCDPER Members

Identification of a Pyranocoumarin Photosensitizer that is a Potent Inhibitor of Keratinocyte Growth.

Tue, 01/23/2018 - 03:00

Related Articles Identification of a Pyranocoumarin Photosensitizer that is a Potent Inhibitor of Keratinocyte Growth. Photochem Photobiol. 2018 Jan 06;: Authors: Laskin JD, Jan YH, Jetter MM, Guillon CD, Mariano TM, Heck DE, Heindel ND Abstract Photosensitizers are used in the treatment of epidermal proliferation and differentiation disorders such as psoriasis and vitiligo. In the present studies, a ring-expanded carbon homologue of the linear psoralen (furo[3,2-g]benzopyran-7-one) class of photosensitizers, 4,10-dimethyl-2H,8H-benzo[1,2-b:5,4-b']dipyran-2-one (NDH2476), was synthesized and analyzed for biological activity. Following activation by ultraviolet light (UVA, 320-400 nm), NDH2476 was found to be a potent inhibitor of keratinocyte growth (IC50 = 9 nM). Similar derivatives methylated in the pyrane ring, or containing a saturated pyrane ring structure, were markedly less active or inactive as photosensitizers. NDH2476 was found to intercalate and damage DNA following UVA light treatment as determined by plasmid DNA unwinding and nicking experiments. Taken together, these data demonstrate that an intact furan ring in psoralen photosensitizers is not required for keratinocyte growth inhibition or DNA damage. Our findings that low nanomolar concentrations of a benzopyranone derivative was active as photosensitizer indicates that this or a structurally related compound may be useful in the treatment of skin diseases involving aberrant epidermal cell growth and differentiation. This article is protected by copyright. All rights reserved. PMID: 29315592 [PubMed - as supplied by publisher]

Categories: Publications from UCDPER Members

Sulfur mustard induced mast cell degranulation in mouse skin is inhibited by a novel anti-inflammatory and anticholinergic bifunctional prodrug.

Tue, 01/09/2018 - 15:00

Related Articles Sulfur mustard induced mast cell degranulation in mouse skin is inhibited by a novel anti-inflammatory and anticholinergic bifunctional prodrug. Toxicol Lett. 2017 Nov 07;: Authors: Joseph LB, Composto GM, Perez RM, Kim HD, Casillas RP, Heindel ND, Young SC, Lacey CJ, Saxena J, Guillon CD, Croutch CR, Laskin JD, Heck DE Abstract Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent known to cause skin inflammation, necrosis and blistering. Evidence suggests that inflammatory cells and mediators that they generate are important in the pathogenic responses to SM. In the present studies we investigated the role of mast cells in SM-induced skin injury using a murine vapor cup exposure model. Mast cells, identified by toluidine blue staining, were localized in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction. In control mice, 48-61% of mast cells were degranulated. SM exposure (1.4g/m(3) in air for 6min) resulted in increased numbers of degranulated mast cells 1-14days post-exposure. Treatment of mice topically with an indomethacin choline bioisostere containing prodrug linked by an aromatic ester-carbonate that targets cyclooxygenases (COX) enzymes and acetylcholinesterase (1% in an ointment) 1-14days after SM reduced skin inflammation and injury and enhanced tissue repair. This was associated with a decrease in mast cell degranulation from 90% to 49% 1-3days post SM, and from 84% to 44% 7-14days post SM. These data suggest that reduced inflammation and injury in response to the bifunctional indomethacin prodrug may be due, at least in part, to abrogating mast cell degranulation. The use of inhibitors of mast cell degranulation may be an effective strategy for mitigating skin injury induced by SM. PMID: 29127031 [PubMed - as supplied by publisher]

Categories: Publications from UCDPER Members

Diacetyl/l-Xylulose Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells.

Sun, 11/12/2017 - 03:00

Related Articles Diacetyl/l-Xylulose Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells. Chem Res Toxicol. 2017 Jul 17;30(7):1406-1418 Authors: Yang S, Jan YH, Mishin V, Heck DE, Laskin DL, Laskin JD Abstract Reactive carbonyls such as diacetyl (2,3-butanedione) and 2,3-pentanedione in tobacco and many food and consumer products are known to cause severe respiratory diseases. Many of these chemicals are detoxified by carbonyl reductases in the lung, in particular, dicarbonyl/l-xylulose reductase (DCXR), a multifunctional enzyme important in glucose metabolism. DCXR is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Using recombinant human enzyme, we discovered that DCXR mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity preferentially utilized NADH as a cosubstrate and was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione). Using 9,10-phenanthrenequinone as the substrate, quinone redox cycling was found to inhibit DCXR reduction of l-xylulose and diacetyl. Competitive inhibition of enzyme activity by the quinone was observed with respect to diacetyl (Ki = 190 μM) and l-xylulose (Ki = 940 μM). Abundant DCXR activity was identified in A549 lung epithelial cells when diacetyl was used as a substrate. Quinones inhibited reduction of this dicarbonyl, causing an accumulation of diacetyl in the cells and culture medium and a decrease in acetoin, the reduced product of diacetyl. The identification of DCXR as an enzyme activity mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. These activities, together with the inhibition of dicarbonyl/l-xylulose metabolism by redox-active chemicals, as well as consequent deficiencies in pentose metabolism, are likely to contribute to lung injury following exposure to dicarbonyls and quinones. PMID: 28595002 [PubMed - indexed for MEDLINE]

Categories: Publications from UCDPER Members