The Dihydroxy Metabolite of the Teratogen Thalidomide Causes

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The Dihydroxy Metabolite of the Teratogen Thalidomide Causes Oxidative DNA Damage Tasaduq Hussain Wani, Anindita Chakrabarty, Norio Shibata, Hiroshi Yamazaki, F. Peter Guengerich, and Goutam Chowdhury Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00127 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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Chemical Research in Toxicology

The Dihydroxy Metabolite of the Teratogen Thalidomide Causes Oxidative DNA Damage Tasaduq H. Wani†, Anindita Chakrabarty†, Norio Shibata‡, Hiroshi Yamazaki§, F. Peter Guengerich¥ and Goutam Chowdhury*†

†

Departments of Chemistry and Life Sciences, SONS, Shiv Nada University, Greater Noida, UP

201314, India ‡

Graduate School of Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555,

Japan §

Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University,

Machida, Tokyo 194-8543, Japan ¥

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee

37232-0146, United States.

*To whom all correspondence should be addressed. Departments of Chemistry, SONS, Shiv Nada University, Greater Noida, UP 201314, India Email: [email protected] Ph: 91-9821131095

The

authors

declare

no

competing

ACS Paragon Plus Environment

financial

interest.


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Mechanism of thalidomide teratogenicity

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Keyword: Thalidomide, teratogenicity, DNA damage, Reactive intermediate

Abstract Thalidomide [α-(N-phthalimido)glutarimide] (1) is a sedative and antiemetic drug originally introduced into the clinic in the 1950s for the treatment of morning sickness. Although marketed as entirely safe, more than 10,000 babies were born with severe birth defects. Thalidomide was banned and subsequently approved for the treatment of multiple myeloma and complications associated with leprosy. Although known for more than 5 decades, the mechanism of teratogenicity remains to be conclusively understood. Various theories have been proposed in the literature including DNA damage and ROS, inhibition of angiogenesis and inhibition of cereblon. All the theories have their merits and limitation. Although the recently proposed cereblon theory has gained wide acceptance, it fails to explain the metabolism and low dose requirement reported by a number of groups. Recently we have provided convincing structural evidence in support of the presences of arene oxide and the quinone reactive intermediates. However, the ability of these reactive intermediates to impart toxicity/teratogenicity needs investigation. Herein we report that the oxidative metabolite of thalidomide, dihydroxythalidomide is responsible for generating ROS and DNA damage. We show using cell lines the formation of comet (DNA damage) and ROS. Using DNA cleavage assays we also show that catalase, radical scavengers and desferal is capable of inhibiting DNA damage. A mechanism of teratogenicity is proposed that not only explains the DNA damaging property but the metabolism, low concentration and species specificity requirements of thalidomide.

2 ACS Paragon Plus Environment

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Wani et. al.

Introduction Thalidomide [α-(N-phthalimido)glutarimide] (TD, 1) is a sedative and antiemetic drug originally introduced in the clinic in the 1950s for the treatment of morning sickness.1 Although marketed as entirely safe, more than 10,000 babies were born between 1957 and 1961 with severe birth defects that resulted in its withdrawal in the early 1960s.1,

2

However, owing to its clinical

properties, TD was approved in 1998 for the treatment of lesions associated with leprosy and in 2006 for multiple myeloma.3, 4 In addition, TD is being tested for the treatment of many diseases including refractory esophageal Crohn’s disease, recurrent bleeding resulting from gastric angiodysplasia and hereditary hemorrhagic telangiectasia.5-8 The recent emergence of TD as a drug with clinical potential resulted in renewed interest in both its toxicity and pharmacological mechanisms, none of which are conclusively established. Moreover, prevention of inadvertent exposure of pregnant women to this drug is a continuing challenge, particularly in parts of the world where access to the drug is less restricted. The teratogenicity of TD is very species-specific, being teratogenic in primates and rabbits but not in rats and mice.9 It was initially believed that the R isomer is sedative, whereas the S isomer is teratogenic; however, the two enantiomers are readily interconvertible.10 TD is metabolized

in

the

liver

to

hydroxythalidomide, by P450s.11,

two 12

major

products,

5-hydroxythalidomide

and

5´-

We have previously shown that P450 3A4 and 3A5 also

oxidize TD to the 5-hydroxy and dihydroxy metabolites.13, 14 The second oxidation step in the P450 3A4 pathway generates a reactive intermediate, possibly an arene oxide that can be trapped by the tripeptide glutathione (GSH) to give GSH adducts.3, 15, 16 The observation of GSH adduct formation with 5-hydroxythalidomide was confirmed in vivo in humanized mouse models.13 The dihydroxythalidomide (DHT, 2) product is further oxidized to the potentially toxic quinone 3 ACS Paragon Plus Environment


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intermediate that can also react with GSH to give the corresponding GSH adduct of DHT.17 In both bacterial and AS52 cells thalidomide was found to be not genotoxic.18 Although TD has been known for more than half a century, its mechanism of teratogenicity remains to be convincingly established. More than 30 theories have been proposed including generation of reactive oxygen species (ROS), inhibition of angiogenesis, and non-covalent binding to the protein cereblon.2,

19-22

However, none of the proposed theories are without

limitations. For example, the ROS theory does not indicate the species responsible for ROS generation or the mechanism of ROS formation;22 the anti-angiogenesis theory proposes the involvement of a reactive intermediate but fails to present any direct evidence of any;21 and finally, the cereblon inhibition theory, although has recently gained wide acceptance is unable to explain the metabolism and low dose requirements (concentration of TD that is significantly higher than clinically relevant exposure levels were used in some of the reported experiments).2327

Interestingly, the need for metabolism of TD for teratogenicity is consistently reported in the

literature.20, 28 Recently, we provided direct evidence for the presence of arene oxide and quinone reactive intermediates, using GSH as a trapping agent.15, 17 However, the ability of these reactive intermediates to impart toxicity/teratogenicity is yet to be demonstrated. Herein we report the DNA damaging properties of the 5,6-dihydroxythalidomide (DHT, 2) metabolite that is readily oxidized to the corresponding quinone.

Experimental Procedures Caution: Thalidomide and its metabolites are extremely teratogenic and toxic. Caution and care should be taken while handling these chemicals. 4 ACS Paragon Plus Environment

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Materials. Unless otherwise mentioned all chemicals used are of the highest quality available and purchased from Sigma Aldrich (St. Louis, MO, USA). Agarose, agar, ethidium bromide, catalase, methanol, ethanol, isopropanol, DMSO, NaCl, EDTA, Tris-HCl, Triton-X 100, NaOH, paraformaldehyde, bovien serum albumin (BSA), biotin, histidine, ampicillin, oxoid nutrient broth No. 2, and protease inhibitor cocktail were obtained from HiMedia (Mumbai, India), sodium fluoride and orthovandate from MP Biomedicals, LLC (Solon Ohio, USA), CellROX Deep Red and Nuc Blue (Hoechst 33342) from ThermoFisher Scientific (Santa Clara, CA), Salmonella typhimurium TA100 from Microbial Type Culture Collection and Gene Bank (MTCC, Chandigarh, India), thalidomide and DMEDA from Tokyo Chemical Industry (Tokyo, Japan), nitrocellulose membranes from MDI Membrane Technologies (Ambala. India), LMPA and LE Agarose from Lonza (Rockland, ME, USA), γ-H2AX Alexa Fluor 488 from Biolegend (San Diego, CA, USA), and PAD-PARP antibody from Abcam (MA, USA). The 293T cell line was purchased from National Centre for Cell Culture (Pune, India) and authenticated by Short Tandem Repeat DNA profiling from Life Code Technology, Delhi, India. HepG2 cells were a generous gift from Dr. Soumya Sinha Roy at the CSIR Institute of Genomics and Integrative Biology, New Delhi. HUVEC was procured from HiMedia (Mumbai, India).

DNA Cleavage Assays. In a typical DNA cleavage reaction, plasmid DNA (pUC19, 1 µg) was incubated with dihydroxy thalidomide (DHT, 0.5-25 µM) in potassium phosphate buffer (1 mM, pH 7.4) at 37 ˚C for 12 h. For reactions containing an NADPH-generating system, 1 µl of it was added such that the final concentration of NADP+ is 250 µM. To prepare the NADPH-generating system, equal volumes of NADP+ (10 mM), and glucose 6-phosphate (100 mM) were premixed and glucose 6-phosphate dehydrogenase (1U) was added to it. Following incubations, reactions 5 ACS Paragon Plus Environment


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were subjected to DMEDA (100 mM) workup for 2 hours at 37 ˚C, quenched by addition of 5 µL of glycerol loading buffer, and then electrophoresed for 45 min at 80 V in 1% agarose gel (w/v) containing 0.5 µg/mL ethidium bromide. DNA was visualized and quantified using an AlphaImager (ProteinSimple, CA) gel documentation system. Strand breaks per plasmid DNA molecule (S) were calculated using the equation S=-ln f1, where f1 is the fraction of plasmid present as form I. For assays with radical scavengers, methanol (1 M), isopropanol (1 M), or DMSO (1 M) was used. For some reactions catalase was used at concentration of 250 µg/mL, ferrous sulfate at 010 mM or desferal at 1 mM.

Comet Assay. In a typical assay, sub-confluent cells treated with DHT, thalidomide, 5hydroxythalidomide or menadione (10 µM each) for 1.5, 3, or 12 h were sandwiched in 0.5% low melting agarose on pre-coated agarose (1%) slides. Sandwiched cells were incubated in lysis buffer (10 mM Tris-HCl buffer (pH 10) containing 2.5 M NaCl, 100 mM EDTA, and 1% TritonX-100 (v/v)) for 2 hours, followed by 30 min incubation in alkaline buffer (300 mM NaOH and 1 mM EDTA, pH>13). Slides were electrophoresed in the same buffer at 21 V/300 mA for 30 min. Neutralization was done in 400 mM Tris-HCl (pH 7.5) buffer and staining with 1µg/mL ethidium bromide. All steps were carried out at 4 °C. Fixing was done with absolute ethanol, and imaging was done using a Leica DFC450C microscope (Wetzlar, Germany). ImageJ plugin OpenComet software was used to quantify the DNA damage.29

Reactive Oxygen Species Detection. Sub-confluent cells were initially treated with 10 µM of DHT for 1.5 and 3 h, and then CellROX Deep Red (5 µM) reagent was added and incubated for 6 ACS Paragon Plus Environment

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30 min. Following incubation cells were treated with Hoechst counter stain for another 10 min, washed with phosphate-buffered saline solution, and immediately taken for imaging. For a positive control, menadione was used at a concentration of 100 µM for 2 h.

Western Blot Analysis for PAR-PARP. Cells were treated with 10 µM DHT for 15, 45, 90, and 180 min and subsequently lysed using RIPA Lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0 and NP40 1%). Protease inhibitor cocktail along with NaF (500 mM) and orthovandate (100 mM) were added to the lysate and incubated on ice for 30 min, with a single brief vortex mixing of 5 s. After incubation, the lysate was centrifuged at 1,000 g for 10 min and supernatant taken. Quantification of protein was done using a Bradford assay.30 Proteins in the lysate were separated using 8% SDS-PAGE and transferred to a nitrocellulose membrane. PAD-PARP (Abcam, 1:500, v/v) was used to check for DNA damage.31 Equal loading was confirmed using

β-actin. Imaging was done on a LICOR instrument.

Statistical Analysis. All data were analyzed using GraphPad Prism software (Prism 5.03). Data are expressed as mean ±SD or mean ±SEM. A P-value of

The Dihydroxy Metabolite of the Teratogen Thalidomide Causes

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