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. Author manuscript; available in PMC: 2025 Sep 16.
Published in final edited form as: Biochem Pharmacol. 2018 Jul 18;155:455–467. doi: 10.1016/j.bcp.2018.07.016

Effects of human sulfotransferases on the cytotoxicity of 12-hydroxynevirapine☆

Jia-Long Fang 1,*, Lucie Loukotková 1, Priyanka Chitranshi 1, Gonçalo Gamboa da Costa 1, Frederick A Beland 1
PMCID: PMC12434715  NIHMSID: NIHMS2110485  PMID: 30028994

Abstract

Nevirapine, a non-nucleoside reverse transcriptase inhibitor used for the treatment of AIDS, can cause serious skin rashes and hepatotoxicity. Previous studies have indicated that the benzylic sulfate 12-sulfoxynevirapine, the formation of which is catalyzed by human sulfotransferases (SULTs), may play a causative role in these toxicities. To characterize better the role of 12-sulfoxynevirapine in nevirapine-induced cytotoxicity, the ability of 12 expressed human SULT isoforms to conjugate 12-hydroxynevirapine was assessed. Of the 12 human SULTs, no detectable 12-sulfoxynevirapine was observed with SULT1A3, SULT1C2, SULT1C3, SULT2B1, SULT4A1, or SULT6B1. As determined by the Vmax/Km ratio, SULT2A1 had the highest overall 12-hydoxynevirapine sulfonation activity; lower activities were observed with SULT1A1, SULT1A2, SULT1B1, SULT1C4, and SULT1E1. Incubation of 12-sulfoxynevirapine with glutathione and cysteine led to adduct formation; lower yields were obtained with deoxynucleosides. 12-Hydroxynevirapine was more cytotoxic than nevirapine to TK6, TK6/SULT vector, and TK6/SULT2A1 cells. With nevirapine, there was no difference in cytotoxicity among the three cell lines, whereas with 12-hydroxynevirapine, TK6/SULT2A1 cells were more resistant than TK6 and TK6/SULT vector cells. Co-incubation of 12-hydroxynevirapine with the competitive SULT2A1 substrate dehydroepiandrosterone decreased the level of 12-sulfoxynevirapine and increased the cytotoxicity in TK6/SULT2A1 cells. These data demonstrate that although 12-sulfoxynevirapine reacts with nucleophiles to form adducts, sulfonation of 12-hydroxynevirapine decreases the cytotoxicity of 12-hydroxynevirapine in TK6 cells.

Keywords: Nevirapine, Sulfonation, Cytotoxicity, Adducts

1. Introduction

Nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido [3,2-b:2′,3′-e][1,4]diazepin-6-one, Fig. 1) is a non-nucleoside reverse transcriptase inhibitor used for the treatment of acquired immunodeficiency syndrome and the prevention of the mother-to-child transmission of human immunodeficiency virus-1 [1,2]. Although nevirapine currently remains one of the most prescribed antiretroviral drugs in the developing countries, it can cause skin rashes and hepatotoxicity [3–6]. As a consequence, the US Food and Drug Administration placed a black box warning label for nevirapine-induced life-threatening hepatotoxicity and skin reactions. The reasons for the adverse effects of nevirapine treatment are currently not understood; however, oxidative metabolism of nevirapine to reactive metabolites capable of reacting with nucleophiles (protein and/or DNA) may be involved in the initiation of toxic responses.

Fig. 1.

Fig. 1.

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Chemical structures of nevirapine, 12-hydroxynevirapine, and 12-sulfoxynevirapine.

Several studies have demonstrated that nevirapine undergoes significant metabolism in both humans and experimental animals [7–13]. The oxidative metabolism of nevirapine is mediated mainly by the cytochrome P450 (CYP) isozymes CYP3A, CYP2D6, and CYP2B6 [4,14]. The major hydroxylated metabolites are 2-, 3-, 8-, and 12-hydroxynevirapine; another major metabolite is 4-carboxynevirapine, which is formed by the further oxidation of 12-hydroxynevirapine. Subsequent sulfonation of 12-hydroxynevirapine by a family of cytosolic sulfotransferases (SULTs) produces a potentially reactive benzylic-like sulfate. Recently, two studies have reported the presence of 12-sulfoxynevirapine in the urine and bile of treated rats [8,15], and several studies have demonstrated that 12-mesyloxynevirapine, a surrogate for 12-sulfoxynevirapine, forms multiple adducts with deoxynucleosides, salmon testis DNA, amino acids, peptides, human serum albumin, and hemoglobin in vitro [16–19]. In addition, nevirapine-protein adducts have been detected in patients administered therapeutic doses of nevirapine [20,21]. Although a mesylate is a much better leaving group than a sulfate, it is not formed in vivo, and the role of 12-sulfoxynevirapine in the formation of DNA and/or protein adducts in vivo is not clear.

In human tissues, SULTs catalyze the sulfonation of a multitude of xenobiotics, many therapeutic drugs, and a variety of endogenous compounds, including steroids, thyroid hormones, and monoamine neurotransmitters [22,23]. The SULT isoforms involved in the sulfonation of 12-hydroxynevirapine have not been described. In this study, we investigated the ability of 12 human SULT isoforms to conjugate 12-hydroxynevirapine, the reactivity of 12-sulfoxynevirapine with deoxynucleosides, cysteine, and glutathione, and the effect of human SULT2A1-mediated metabolism of 12-hydroxynevirapine on cell growth. The results indicate that several of human SULTs are involved in the sulfonation of 12-hydroxynevirapine, with SULT2A1 being the predominant isoform. Covalent adducts were formed in the reactions of 12-sulfoxynevirapine with deoxynucleosides, glutathione, and cysteine, with the reactivity being much lower towards deoxynucleosides than towards glutathione or cysteine. Although 12-sulfoxynevirapine was nucleophilic, sulfonation of 12-hydroxynevirapine decreased its cytotoxicity in human lymphoblastoid TK6 cells.

2. Materials and methods

2.1. Chemicals and reagents

Nevirapine (Fig. 1) was obtained from Cipla Ltd. (Mumbai, India). 12-Hydroxynevirapine was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Ammonium acetate, anhydrous pyridine, 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol (Bis-Tris), cysteine, 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), dimethyl sulfoxide (DMSO), dehydroepiandrosterone (DHEA), ethylenediaminetetraacetic acid (EDTA), glutathione, RPMI 1640 medium, pentachlorophenol, sulfur trioxide pyridine complex, thymidine (T), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). Acetonitrile, Dulbecco’s Modified Eagle Medium (DMEM), methanol, penicillin–-streptomycin solution, puromycin, 2.5% trypsin, and the BCA Protein Assay kit were purchased from Thermo Fisher Scientific, Inc. (Pittsburgh, PA). Adenosine 3′-phosphate 5′-phosphosulfate lithium salt hydrate (PAPS), and mouse monoclonal antibodies to human SULT2A1 and β-actin were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). HPLC-UV analysis at 254 nm conducted in our laboratory indicated that the purity of PAPS was 79%; adenosine 3′-phosphate 5′-phosphate was not detected. Fetal bovine serum (FBS) was acquired from Atlanta Biologicals (Lawrenceville, GA). [35S]PAPS (specific activity 2.4 Ci/mmol, purity > 99.0%) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO).

2.2. Synthesis and characterization of 12-sulfoxynevirapine

12-Sulfoxynevirapine was synthesized by reacting 6 mg 12-hydroxynevirapine in 250 μl anhydrous pyridine with 75 mg sulfur trioxide pyridine complex using an Eppendorf Thermomixer R (Eppendorf North America, Hauppauge, NY) at 60 °C, with shaking at 1000 rpm, for 4 h. The 12-hydroxynevirapine and sulfur trioxide pyridine complex had previously been dried in the presence of phosphorus pentoxide under vacuum for 2 days. After the reaction, the mixture was diluted (1:200) with methanol and the resulting solution was subjected to analysis by reversed-phase high-performance liquid chromatography (HPLC) using a Waters HPLC system consisting of a 600 Controller, a 996 Photodiode Array detector, and a 717 Plus autosampler (Waters Corporation, Milford, MA). Samples were injected onto a 4.6 × 250 mm C18 (5 μm particle size) Luna column (Phenomenex, Torrance, CA). HPLC separations were performed with acetonitrile (solvent A) and 50 mM ammonium acetate, pH 5.0 (solvent B) as follows: 5 min, 90% solvent B; 5–35 min, linear gradient to 80% solvent B; 36–47 min, linear gradient to 0% solvent B. The HPLC flow rate was 1 ml/min. The column was washed with 100% solvent A for 15 min and equilibrated for 15 min with 90% solvent B after every run. When [35S]12-sulfoxynevirapine was measured, the HPLC system included a β-RAM radiochemical detector (IN/US, Tampa, FL), with Ultima-Flo M scintillation fluid (PerkinElmer, Waltham, MA) at a flow rate of 2 ml/min.

The peak corresponding to 12-sulfoxynevirapine was tentatively identified by relative retention time. The peak eluting from the HPLC column was collected, purified, and characterized by mass spectrometry system I (described in the following section) and 1H NMR spectroscopy. Aliquots of the synthesized standard were used as a UV standard for identification of 12-sulfoxynevirapine.

2.3. Mass spectrometry and 1H NMR analyses

Mass spectrometry system I was a Waters Acquity ultra-performance liquid chromatography (UPLC) system coupled with a Waters Quattro Premier XE tandem quadrupole mass spectrometer equipped with an electrospray ionization source (ESI-MS/MS). Chromatographic separations were achieved on a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm particle size) maintained at 30 °C using the following gradient between solvent A (water containing 10% acetonitrile and 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid): 0–2.5 min 0–50% B, followed by column equilibration to initial conditions (0% B) at a flow rate of 500 μl/min. The mass spectrometer was operated in the negative electrospray ionization mode. The collision gas was argon at a flow rate of 350 μl/min and the collision energy was optimized to provide an optimal fragmentation of the analyte.

Mass spectrometry system II was a Waters ACQUITY I-class UPLC system coupled to a Waters Xevo TQ-S tandem quadrupole mass spectrometer operated in the positive electrospray ionization mode. The chromatographic separation was performed on a Waters BEH C18 column (2.1 × 150 mm, 1.7 μm) maintained at 30 °C using a 30-min linear gradient of 5 to 90% acetonitrile in water containing 0.1% formic acid. The flow rate was 200 μl/min.

1H NMR spectroscopy was conducted with a Bruker Avance III spectrometer equipped with a Bruker BBFO Plus Smart Probe at 500 MHz and 300 K (Bruker Instruments, Billerica, MA).

2.4. Cell culture

The human embryonic kidney cell line HEK293 and lymphoblastoid cell line TK6 were obtained from American Type Culture Collection (ATCC, Manassas, VA). HEK293 cell lines overexpressing SULTs were prepared as described in Fang et al. [24]. HEK293 cell lines were cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin solution. TK6 cells were cultured in suspension in RPMI 1640 supplemented with 10% FBS and penicillin-streptomycin solution. All cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO2.

2.5. Preparation of cytosol from mouse liver, HEK293 cells, and HEK293/SULT-overexpressing cell lines

Frozen livers, pooled from 10 male B6C3F1 mice (10 weeks old, Charles River Laboratories, Wilmington, NC), were homogenized in ice-cold 5 mM Bis-Tris (pH 7.0) and 0.1 mM EDTA. The homogenate was centrifuged at 10,000g for 10 min at 4 °C to remove debris and large organelles, and then the supernatant was centrifuged at 100,000g for 60 min at 4 °C. The resulting supernatant fraction (cytosol) was collected and the protein concentration was determined using a BCA Protein Assay kit.

Cytosols were also prepared from 12 HEK293/SULT-overexpressing cell lines expressing individual human SULT isoforms (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C3, SULT1C4, SULT1E1, SULT2A1, SULT2B1, SULT4A1, and SULT6B1) and HEK293/SULT vector cells [24]. For each cell line, approximately 2 × 108 cells were harvested, lysed in 5 mM Bis-Tris (pH 7.0) and 0.1 mM EDTA for 30 min on ice, and sonicated three times for 10 s each. The homogenate was centrifuged and the protein content of the cytosol was determined as described for the mouse liver samples.

2.6. Analysis of sulfonation activity

The sulfonation activity of pooled human or Sprague-Dawley rat liver cytosols (Corning Life Sciences, Tewksbury, MA), or cytosols prepared from the B6C3F1 mouse livers or HEK293/SULT-overexpressing cell lines towards 12-hydroxynevirapine was assayed in a 125 μl final reaction volume by incubating 1.0 mM 12-hydroxynevirapine with cytosols (0.5–1.8 mg of cytosol protein), 2 mM EDTA, 500 μM PAPS, and 100 mM sodium phosphate (pH 7.2) at 37 °C for 1 h with gentle shaking. An initial sulfonation activity screening was performed with a radiochemical assay using 500 μM [35S]PAPS (diluted with unlabeled PAPS to a specific activity of 19.8 mCi/mmol), with the assay conditions optimized (the concentration of PAPS, amount of cytosol proteins, and incubation time) to maximize the level of detection within a linear range. Kinetic analyses for SULTs exhibiting 12-hydroxynevirapine sulfonation activity were performed as described above, using an incubation time of 1 h. Sulfonation was linear with each SULT isoform tested for a period of at least 3 h. The Km and Vmax for the sulfonation of 12-hydroxynevirapine by individual SULT isoforms were calculated using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA). Negative controls for the sulfonation reactions included incubations with cytosols from HEK293 and HEK293/SULT vector cells and incubations conducted in the absence of 12-hydroxynevirapine or PAPS. Reactions were terminated by the addition of an equal volume of ice-cold methanol. Precipitated material was removed by centrifugation (5 min, 14,000g), and the supernatants (200 μl) were analyzed for 12-sulfoxynevirapine using the reversed-phase HPLC assay described as above. The lower limit of quantification for 12-sulfoxynevirapine was 10 pmol, based on radiochemical detection and quantification of radioactivity within the 12-sulfoxynevirapine HPLC peak as determined with the IN/US β-RAM radioactivity detection program. The intraday and interday precision variation (coefficient of variation) was < 10%.

2.7. Reaction of nevirapine, 12-hydroxynevirapine, or 12-sulfoxynevirapine with dA, dG, dC, or T

Solutions of nevirapine (15 mM), 12-hydroxynevirapine (15 mM), or 12-sulfoxynevirapine (15 mM) were incubated with dG, dA, dC, or T (each 15 mM) in 400 μl of 50 mM Bis-Tris (pH 7.1) and 0.1 mM EDTA at 37 °C with shaking for up to 15 days. Adducts formed with the deoxynucleosides were extracted from the reaction mixtures by adding 20 μl 1 M ammonium bicarbonate and 100 μl n-butanol. Upon vortexing for 1 min and centrifugation at 21,000g for 8 min, the n-butanol layer was collected, evaporated under speed vacuum, reconstituted in the initial mobile phase composition (water/acetonitrile/formic acid 95/5/0.1 (v/v/v)), and characterized by mass spectrometry system II. The chromatographic separations were achieved on a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm particle size), maintained at 30 °C, using a 30-min linear gradient of 5–90% acetonitrile in water containing 0.1% formic acid. The flow rate was 200 μl/min.

2.8. Reaction of nevirapine, 12-hydroxynevirapine, or 12-sulfoxynevirapine with cysteine or glutathione

Solutions of nevirapine (15 mM), 12-hydroxynevirapine (15 mM)), or 12-sulfoxynevirapine (15 mM) were incubated with cysteine (15 mM) or glutathione (15 mM) in 400 μl of 100 mM sodium phosphate (pH 7.2) at room temperature with shaking for 1, 2, 6, and 24 h. The resulting conjugates were analyzed by reversed-phase HPLC assay as above, purified, and characterized by mass spectrometry system II using a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm particle size), maintained at 40 °C, with a 1.5 min linear gradient of 5–50% acetonitrile in water containing 0.1% formic acid. The flow rate was 400 μl/min.

2.9. Generation of TK6 cells overexpressing human SULT2A1

Previous data indicated basal levels of human SULTs in HEK293 cells [24], whereas TK6 cells did not show any detectable sulfonation activity, using triclosan as the substrate. To examine the effects of SULT2A1-mediated metabolism on the toxicity of 12-hydroxynevirapine, a stable TK6 cell line that overexpresses human SULT2A1 was generated as described previously [24]. Forty-eight hours post-transfection, the cells were passaged and subsequently grown in media containing 2 μg/ml puromycin for the selection of puromycin-resistant cells that stably expressed human SULT2A1 (TK6/SULT2A1 cells) or the mock vector (TK6/SULT vector cells).

When the cells reached 80–90% confluence, they were harvested by centrifugation and washed three times in phosphate-buffered saline (PBS). Cytosols were prepared as described above. The protein concentrations of the cytosol preparation were determined using a BCA Protein Assay kit.

2.10. Western blot analysis

The levels of human SULT2A1 protein in TK6/SULT2A1 cells were measured by Western blot. Aliquots of cytosolic proteins was separated by SDS polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against human SULT2A1 (1:1000) or β-actin (1:2000), followed by a secondary antibody conjugated to horseradish peroxidase. The blots were then detected by chemiluminescence using Immobilon Western Horseradish Peroxidase Substrate (Millipore Corporation, Billerica, MA), a UVP BioSpectrum AC Imaging System, and VisionWorks LSD Image Acquisition & Analysis Software (UVP LLC, Upland, CA).

2.11. Measurement of the half inhibitory concentration (IC50) of nevirapine and 12-hydroxynevirapine on cell growth

The IC50 values of nevirapine and 12-hydroxynevirapine in TK6, TK6/SULT vector, and TK6/SULT2A1 cells were assessed using an MTT assay. Cells were seeded at a density of 2.5 × 104 cells/ml in 24-well plates with a volume of 1 ml/well and cultured for 24 h prior to treatment with various concentrations of nevirapine (12.5–400 μM) or 12-hydroxynevirapine (6.25–150 μM) for 48 h. Control cells were fed with complete culture medium containing 0.1% (v/v) DMSO, which had no effect on cell growth. At the end of the treatment, 100 μl of 5 mg/ml MTT solution was added to each well and incubated for 4 h at 37 °C. The resulting formazan was dissolved in 1 ml of 10% sodium dodecyl sulfate (SDS)/0.01 N HCl solution and the absorbance at 570 nm was determined with a BioTek Cytation 5 cell imaging multi-mode reader. The IC50 values were obtained from the cell growth curves, using GraphPad Prism 6.0.

2.12. The effect of sulfonation on the cytotoxicity of 12-hydroxynevirapine in TK6/SULT2A1 cells

TK6/SULT2A1 and TK6/SULT vector cells were treated with an IC50 concentration of 12-hydroxynevirapine (i.e. 90 μM and 45 μM for TK6/SULT2A1 and TK6/SULT vector cells, respectively) in the presence or absence of the competitive SULT substrates DHEA (5 μM) and pentachlorophenol (10 μM) for 48 h. The cytotoxicity was assessed using an MTT assay as described above.

2.13. Data analysis

Data are presented as the mean ± standard deviation of three independent experiments. Comparisons amongst concentrations were conducted by one-way analysis of variance, with pairwise comparisons versus control group being performed by Dunnett’s method. When necessary, the data were log-transformed to maintain an equal variance or normal data distribution. The results were considered significant at p < 0.05.

3. Results

3.1. Characterization of 12-sulfoxynevirapine

The reaction of 12-hydroxynevirapine in anhydrous pyridine with sulfur trioxide pyridine complex led to the formation of a peak at 24.5 min (Fig. 2A). The reaction was quantitative, with all the 12-hydroxynevirapine being converted to 12-sulfoxynevirapine, as determined by reversed-phase HPLC analysis. The peak was collected, purified, and subjected to structural characterization by mass spectrometry system I and 1H NMR spectroscopy.

Fig. 2.

Fig. 2.

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HPLC analysis of 12-sulfoxynevirapine. 12-Sulfoxynevirapine formation in reaction mixture (A) and the 12-hydroxynevirapine standard (B) were detected by UV at 280 nm. (C) ESI-mass spectrum of 12-sulfoxynevirapine at a collision energy of 25 eV. The fragmentation indicated sulfonation of the hydroxyl group of 12-hydroxynevirapine.

A full scan analysis of the purified peak in the negative electrospray ionization mode revealed the presence of a prominent peak in the mass chromatogram with an m/z = 361, consistent with the expected molecular weight of an anion of sulfoxynevirapine. Daughter scan analysis of this ion at an optimized collision energy of 25 eV revealed the formation of fragment ions corresponding to the loss of -SO3 (m/z 281) and -CH2OSO3 (m/z 251), providing clear evidence that the sulfonation of 12-hydroxynevirapine occurred at its hydroxyl group (Fig. 2C).

The structural characterization by 1H NMR was carried out using the purified peak. The 1H NMR spectrum was consistent with the structure of 12-sulfoxynevirapine. Of note, the two protons in the C12 methylene group afforded two doublets with a germinal coupling constant of 14.5 Hz, evidence of magnetic anisotropy in the moiety. This anisotropy may be due to the relative bulkiness of the sulfoxy group and possible intramolecular stabilization of this group by hydrogen bonding with the contiguous amide proton. The NMR data (chemical shifts are reported in ppm, and the coupling constants are reported in Hz) are as follows: 1H NMR (DMSO-d6): δ 8.51 (H9, 1H, dd, Jortho = 4.8, Jmeta = 1.9), 8.20 (H2, 1H, d, Jortho = 5), 8.02 (H7, 1H, dd, Jortho = 7.6, Jmeta = 2.0), 7.22–7.18 (H8, H3, 2H, m), 5.09 (H12a, 1H, d, Jgem = 14.5), 4.80 (H12b, 1H, d, Jgem = 14.5), 3.63 (H13, 1H, m), 0.89–0.87 (H14, H15, 2H, m), 0.38–0.34 (H14, H15, 2H, m).

Stability studies conducted with 12-sulfoxynevirapine using reversed-phase HPLC analyses, as described above, indicated that 12-sulfoxynevirapine was stable in either 50 mM Bis-Tris, pH 7.1 or 100 mM sodium phosphate, pH 7.2 at room temperature up to 2 months.

3.2. Sulfonation of 12-hydroxynevirapine with cytosols from SULT-overexpressing cells

To elucidate whether any human SULTs exhibited activity towards 12-hydroxynevirapine, a comprehensive screening of known human SULTs was conducted. The cell lines overexpressing individual SULT isoforms were constructed previously [24]. Cytosols prepared from livers of humans, rats, and mice, as well as cytosols prepared from SULT-overexpressing cells showed sulfonation activity toward the positive control substrate triclosan (Table 1). As summarized in Table 1, cytosols from human and rat liver and cells overexpressing SULT1A1, SULT1A2, SULT1B1, SULT1C4, SULT1E1, and SULT2A1 exhibited detectable levels of 12-hydroxynevirapine sulfonation activity. No 12-sulfoxynevirapine was observed for cytosols from mouse liver or from cells overexpressing SULT1A3, SULT1C2, SULT1C3, SULT2B1, SULT4A1, or SULT6B1. HPLC chromatograms for the analysis of 12-sulfoxynevirapine from HEK293/SULT2A1 and HEK293 cytosols are shown in Fig. 3. Incubation of 12-hydroxynevirapine with HEK293/SULT2A1 cytosols at 37 °C for 1 h led to the formation of a peak (Fig. 3A) exhibiting a retention time identical to that obtained for the 12-sulfoxynevirapine UV standard (Fig. 2A). Radiochemical detection confirmed the formation of [35S]12-sulfoxynevirapine from [35S] PAPS in the same cytosols (Fig. 3B). No such metabolite was detected in reactions using HEK293 cytosols, HEK293/SULT vector cytosols (Fig. 3C and 3D), or in incubations lacking 12-hydroxynevirapine or PAPS.

Table 1.

Sulfonation of triclosan and 12-hydroxynevirapine by liver cytosols and individual human SULTs.

pmol min−1 mg cytosolic protein−1
Triclosana 12-Hydroxynevirapineb
Rat liver 527 ± 96.4 298 ± 7.7
Human liver 224 ± 39.9 55.7 ± 3.4
Mouse liver 300 ± 3.5 n.d.
SULT1A1 327 ± 21.6 47.7 ± 0.7
SULT1A2 511 ± 11.6 7.7 ± 0.8
SULT1A3 477 ± 46.2 n.d.
SULT1B1 533 ± 21.6 6.1 ± 0.1
SULT1C2 28.5 ± 0.2 n.d.
SULT1C3 103 ± 11.1 n.d.
SULT1C4 504 ± 13.1 27.4 ± 7.1
SULT1E1 495 ± 47.8 25.3 ± 1.4
SULT2A1 100 ± 0.3 197 ± 8.5
SULT2B1 46.6 ± 1.9 n.d.
SULT4A1 50.4 ± 0.6 n.d.
SULT6B1 33.8 ± 1.0 n.d.
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a

Sulfonation assays were conducted in a 125 μl final reaction volume by incubating 250 μM [2,4-dichlorophenyl-14C(U)]triclosan (specific activity: 35.6 mCi/mmol) with 0.5 mg cytosolic protein, 500 μM PAPS, and 100 mM sodium phosphate, pH 7.2, at 37 °C for 2 h. Reactions were terminated by the addition of an equal volume of ice-cold methanol. Precipitated material was removed by centrifugation at 14,000g at 4 °C for 30 min and the supernatants were analyzed for triclosan sulfate by a reversed-phase HPLC with radiochemical detection as previously described (Fang et al., 2016).

b

Sulfonation assays using 1.0 mM 12-hydroxynevirapine at 37 °C for 1 h, as described in Materials and Methods. n.d.: not detected.

Fig. 3.

Fig. 3.

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HPLC analysis of 12-sulfoxynevirapine formation in cytosols from individual SULT-overexpressing cells. Shown are incubations of 12-hydroxynevirapine with 500 μg of cytosolic protein from HEK293/SULT2A1 cells (A and B) and 1.8 mg of cytosolic protein from HEK293/vector cells (C and D). (A) and (C) 12-sulfoxynevirapine formation as assessed by UV at 280 nm; (B) and (D) [35S]12-sulfoxynevirapine formation as assessed by radiochemical detection. Peaks corresponding to [35S]PAPS, 12-hydroxynevirapine, [35S]12-sulfoxynevirapine, and 12-sulfoxynevirapine are indicated by arrows.

The relative enzyme affinities for 12-hydroxynevirapine, as reflected by their apparent Km’s, were SULT1E1 ~ rat liver ~ SULT1C4 > SULT2A1 ~ human liver > SULT1A1 (Table 2). As determined by the Vmax/Km ratio, the order of the overall sulfonation activity towards 12-hydroxynevirapine was rat liver > > SULT2A1 > SULT1E1 > SULT1C4 > human liver ~ SULT1A1 (Table 2). The kinetic parameters of SULT1A2 and SULT1B1 were not determined due to their low sulfonation activity towards 12-hydroxynevirapine.

Table 2.

Kinetic analysis of liver cytosols and individual human SULTs towards 12-hydroxynevirapine.a

Cytosols Kmb
μM		 Vmaxc
pmol min−1 cytosolic
protein mg−1 		Vmax/Km
μl min−1 mg−1
Rat liver 243 ± 93.9 333 ± 38.6 1.4 ± 0.6
Human liver 605 ±116 71.0 ± 4.5 0.12 ± 0.02
SULT1A1 787 ± 62.2 71.2 ± 2.0 0.09 ± 0.01
SULT1C4 260 ± 38.9 56.0 ± 2.2 0.22 ± 0.03
SULT1E1 216 ± 34.0 71.0 ± 2.8 0.33 ± 0.05
SULT2A1 545 ± 68.5 261 ± 12.4 0.48 ± 0.06
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a

All the reactions were performed using liver cytosols or cytosols from individual SULT-overexpressing cell lines. Incubations were for 1 h at 37 °C, as described in Materials and Methods. Kinetic parameters are reported as mean ± s.d. for three individual experiments.

b

Km, apparent Km.

c

Vmax values are adjusted per mg of the corresponding cytosolic protein of individual SULT-overexpressing cell lines.

3.3. Formation of 12-sulfoxynevirapine deoxynucleoside adducts

To investigate the formation of 12-sulfoxynevirapine deoxynucleoside adducts, the reaction of each deoxynucleoside with 12-sulfoxynevirapine and two control reactions of deoxynucleoside and 12-sulfoxynevirapine by themselves were analyzed by mass spectrometry. The adducts were detected only after reaction at 37 °C for 15 days. Based upon the anticipated mass for a protonated adduct resulting from the reaction of 12-sulfoxynevirapine with dA, ion chromatograms at m/z 516 were extracted from the acquired data. Two peaks eluting at 6.61 min and 9.19 min were observed in the reaction (Fig. 4A top panel), but not in the controls. Both peaks provided fragments at m/z 400, stemming from the loss of the deoxyribose moiety [MH+-deoxyribose], and at m/z 265, corresponding to a nevirapine residue (Fig. 4A bottom panel); the minor peak at 6.61 min produced a fragment at m/z 136, which is the MH+ of adenine. The fragmentation of the major adduct is identical to that reported by Antunes et al. [16] from the reaction of 12-mesyloxynevirapine with dA, which supports the hypothesis that the major peak eluting at 9.19 min is 12-(deoxyadenosin-N1-yl)nevirapine. The slightly different fragmentation pattern of the minor peak eluting at 6.61 min suggests that this adduct is the product of a Dimroth rearrangement, as described by Antunes et al. [16], to give 12-(deoxyadenosin-N6-yl)nevirapine.

Fig. 4.

Fig. 4.

Fig. 4.

Fig. 4.

Fig. 4.

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UPLC-MS/MS analysis of the adducts resulting from the reaction of 12-sulfoxynevirapine with deoxynucleosides. The adduct peaks are shaded. (A) Top panel: extracted ion chromatograms of m/z 516 in the reaction of dA with 12-sulfoxynevirapine; bottom panel: the daughter ion spectra of the peak eluting at 9.19 min. (B) Top panel: extracted ion chromatograms of m/z 416 in the reaction of dG with 12-sulfoxynevirapine; bottom panel: the daughter ion spectra of the peak eluting at 7.08 min. (C) Top panel: extracted ion chromatograms of m/z 492 in the reaction of dC with 12-sulfoxynevirapine; bottom panel: the daughter ion spectra of the peak eluting at 6.76 min. (D) Top panel: extracted ion chromatograms of m/z 507 in the reaction of T with 12-sulfoxynevirapine; bottom panel: the daughter ion spectra of the peak eluting at 10.27 min.

Ion chromatograms at m/z 416, the expected mass for a depurinated adduct resulting from the reaction of 12-sulfoxynevirapine with dG, were extracted from the acquired data. As illustrated in Fig. 4B top panel, two peaks were observed at 7.08 and 10.00 min in the reaction of 12-sulfoxynevirapine with dG. A daughter ion scan showed an identical fragmentation pattern for both peaks (Fig. 4B bottom panel). While the formation of N7-substituted dG adducts from a variety of electrophiles followed by depurination is common both in vitro and in vivo [25], reaction at the less nucleophilic and more hindered N9 of guanine is unusual. This evidence indicates that the major adduct eluting at 7.08 min is 12-(guanin-N7-yl)nevirapine and the less abundant adduct eluting at 10.0 min is 12-(guanin-N9-yl)nevirapine, as reported by Antunes et al. [16]. However, to confirm the exact structure of these two adducts, further characterization would be necessary.

A comparison of the control reactions and the reaction of 12-sulfoxynevirapine with dC indicated the formation of three peaks after extraction of m/z 492, the expected mass for the protonated adduct resulting from the reaction of 12-sulfoxynevirapine with dC, from the acquired full scan data (Fig. 4C top panel). A product ion scan indicated that the third peak at 9.38 min (Fig. 4C top panel) was an impurity with the same molecular mass as the adduct. As illustrated in Fig. 4C bottom panel, a characteristic fragment at m/z 376 resulting from loss of the deoxyribose moiety was present in the daughter ion scans of the peak at 6.76 min. Other daughter ions consisted of residues of nevirapine at m/z 265 and cytosine at m/z 112. This fragmentation, which is also in agreement with that published by Antunes et al. [16] from the reaction of 12-mesyloxynevirapine with dC, supports connectivity through N3 of dC (Fig. 4C bottom panel) and thus formation of 12-(deoxycytidin-N3-yl)nevirapine. A similar fragmentation pattern (m/z 376 and 265) of the minor adduct at 7.30 min suggested the formation of 12-(deoxycytidin-N4-yl)nevirapine due to a Dimroth rearrangement of 12-(deoxycytidin-N3-yl)nevirapine.

Two peaks at m/z 507 were observed in the ESI mass chromatogram from the reaction of 12-sulfoxynevirapine with T, but not in the control reactions (Fig. 4D top panel). The major product eluting at 10.27 min displayed a fragment at m/z 391, which is characteristic for loss of the deoxyribose moiety, an additional fragment at m/z 308, and a characteristic nevirapine residue at m/z 265 (Fig. 4D bottom panel). This fragmentation, which is identical to that reported by Antunes et al. [19] from the reaction of 12-mesyloxynevirapine with T, confirms the formation of 12-(thymidin-N3-yl)nevirapine. The mass spectrum of the minor peak eluting at 9.06 min showed characteristic fragments at m/z 391 and 265. This fragmentation pattern and elution prior to the 12-(thymidin-N3-yl)nevirapine adduct are in accordance with published data [19]; thus, the minor peak is presumably the 12-(thymidin-O4-yl) nevirapine adduct.

3.4. Formation of 12-sulfoxynevirapine amino acid adducts

The reactions of 12-sulfoxynevirapine with cysteine or glutathione at room temperature for 1, 2, 6, and 24 h led to the formation one product from each reaction (Fig. 5A and 5B), with the highest yield being observed following 24 h of incubation. The yield of the product from the reaction of 12-sulfoxynevirapine with cysteine and glutathione for 24 h was 5.4 and 1.1%, respectively, based on the peak area. The product peaks were collected, purified, and subjected to structural characterization by mass spectrometry.

Fig. 5.

Fig. 5.

Fig. 5.

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HPLC analysis of 12-(cystein-S-yl)nevirapine and 12-(glutathion-S-yl) nevirapine from the reactions of 12-sulfoxynevirapine with cysteine (A) or glutathione (B) as described in Materials and Methods. (C) Daughter ion spectrum of 12-(cystein-S-yl)nevirapine (m/z 388) at a collision energy of 15 eV and a cone voltage 40 V. (D) Daughter ion spectrum of 12-(glutathion-S-yl) nevirapine (m/z 572) at a collision energy of 20 eV and a cone voltage 40 V. The acquired fragments are in accordance with the proposed fragmentation patterns (insets).

Full scan analysis of the purified product from the 12-sulfoxynevirapine reaction with cysteine revealed a molecular ion at m/z 386, which corresponds to the mass of a protonated adduct resulting from the reaction of 12-sulfoxynevirapine with cysteine (Fig. 5C). Tandem mass spectrometry yielded fragments at m/z 341, 299, and m/z 265 (Fig. 5C). Cleavage of the S-C3 bond of the cysteine moiety with sulfur remaining attached to nevirapine resulted in fragment at m/z 299 [nevirapine-SH+H]+. The fragment at m/z 265 resulted from loss of cysteine from the protonated molecule [MH+-cysteine]. The fragmentation pattern shown in Fig. 5C was further supported by previously published data [17] from the reaction of 12-mesyloxynevirapine with N-acetylcysteine, which provides evidence that the product of the reaction is a 12-(cystein-S-yl)nevirapine adduct.

UPLC/MS analysis of the purified product from the 12-sulfoxynevirapine reaction with glutathione confirmed the presence of a protonated adduct at m/z 572. This full scan spectrum is identical to that reported by Antunes et al. [17]. An MS/MS analysis provided fragments at m/z 469 [MH+- C4H8NO2], m/z 443 (as a result of a loss of pyroglutamate), and m/z 299, stemming from cleavage of the cysteine residue in glutathione, with the sulfur remaining attached to the nevirapine moiety [17] (Fig. 5D). Additional fragments included m/z 259 (further loss of the cyclopropyl moiety) and m/z 225 (further loss of H2S). Based on the mass spectral analysis, the reaction product was concluded to be a 12-(glutathion-S-yl)nevirapine adduct.

3.5. Cytotoxicity of nevirapine and 12-hydroxynevirapine in TK6/SULT2A1 cells

An HPLC assay to examine the sulfonation activity among the cytosols from TK6, HEK293, HepG2, and Hep1c1c7 cell lines demonstrated that each of the cell lines, except for TK6 cells, had detectable sulfonation activity towards triclosan [24]. Based upon these results, TK6 cells were selected to construct a cell line overexpressing SULT2A1 (TK6/SULT2A1 cells) that would not be confounded by endogenous sulfonation activity. As shown in Fig. 6A, when assessed by Western blot analysis, SULT2A1 was readily detected in the TK6/SULT2A1 overexpressing cells but not in TK6 and TK6/SULT vector cells. Only cytosols prepared from TK6/SULT2A1 cells showed sulfonation activity towards triclosan (Fig. 6B).

Fig. 6.

Fig. 6.

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(A) Western blotting of human SULT2A1. Cytosolic proteins (40 μg/well) were loaded. β-Actin was used as a loading control. (B) Sulfonation of triclosan by cytosols from TK6, TK6/SULT vector, and TK6/SULT2A1 cells. The results shown are the mean and standard deviation of three independent experiments. n.d.: not detected. (C) The half inhibitory concentration (IC50) of nevirapine and 12-hydroxynevirapine on cell growth. Values in parenthesis are the 95% confidence intervals of the IC50. *Significantly (p < 0.05) different from TK6 and TK6/SULT vector cells; #, significantly (p < 0.05) different from cells exposed to 12-hydroxynevirapine.

To examine the effect of sulfonation on the cytotoxicity of 12-hydroxynevirapine, the cytotoxicity of nevirapine and 12-hydroxynevirapine was compared among TK6, TK6/SULT vector, and TK6/SULT2A1 cells, using an MTT assay. After 48 h of exposure, the IC50 values of nevirapine obtained in the three cell lines were comparable at approximately 130 μM (Fig. 6C). With 12-hydroxynevirapine, the IC50 value in TK6/SULT2A1 cells was 89.9 μM following 48 h exposure. The IC50 value was significantly greater than those at same exposure time in TK6 and TK6/SULT vector cells, which had similar IC50 values (42.6 and 45.1 μM) (Fig. 6C). These data indicate that 12-hydroxynevirapine was more cytotoxic than nevirapine and that sulfonation of 12-hydroxynevirapine decreased the cytotoxicity induced by 12-hydroxynevirapine.

To validate the effects of sulfonation on the cytotoxicity of 12-hydroxynevirapine, TK6/SULT2A1 and TK6/SULT vector cells were exposed to 12-hydroxynevirapine at the concentration equivalent to the IC50 values, in the presence and absence of the competitive SULT2A1 substrates DHEA (5 μM) and pentachlorophenol (10 μM) for 48 h. As shown in Fig. 7A, 12-hydroxynevirapine alone decreased the cell viability to approximately 50% in both cell lines. With TK6/SULT2A1 cells, the addition of DHEA slightly enhanced the cytotoxicity induced by 12-hydroxynevirapine (Fig. 7A top panel), while similar co-treatment did not affect the cytotoxicity induced by 12-hydroxynevirapine in TK6/SULT vector cells (Fig. 7A top panel). DHEA alone was not cytotoxic to both cell lines at the tested concentration. In contrast, co-treatment of TK6/SULT2A1 cells with 12-hydroxynevirapine and pentachlorophenol abolished the cytotoxicity induced by 12-hydroxynevirapine, while co-treatment did not affect the cytotoxicity induced by 12-hydroxynevirapine in TK6/SULT vector cells (Fig. 7A bottom panel). Pentachlorophenol alone was not cytotoxic to both cell lines at the tested concentration. Preliminary experiments indicated that pentachlorophenol was a potent inhibitor of the SULT1E1- and SULT2A1-catalyzed sulfonation of triclosan, with only 44% and 12%, respectively, of the triclosan being sulfated in the presence of 10 μM pentachlorophenol. The co-treatment with 12-hydroxynevirapine and DHEA or pentachlorophenol did not cause changes in the levels of SULT2A1 in TK6/SULT2A1 cells (Fig. 7B). As assayed using HPLC, co-treatment of TK6/SULT2A1 cells with 12-hydroxynevirapine and DHEA or pentachlorophenol for 48 h led to an 18% decrease or a 30% increase in the levels of 12-sulfoxynevirapine, respectively, in the culture media compared to cells treated with 12-hydroxynevirapine only (Fig. 7C). 12-Sulfoxynevirapine was not detected in either TK6/SULT vector cells treated with 12-hydroxynevirapine in the presence or absence of DHEA and pentachlorophenol or in TK6/SULT2A1 cells without 12-hydroxynevirapine. These data clearly indicated that sulfonation attenuates the cytotoxicity of 12-hydroxynevirapine.

Fig. 7.

Fig. 7.

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TK6/SULT2A1 and TK6/SULT vector cells were incubated with an IC50 value of 12-hydroxynevirapine (i.e., 45 and 90 μM, respectively) in the presence and absence of the SULT2A1 substrates DHEA (5 μM) or pentachlorophenol (10 μM) for 48 h. (A) Cell viability was assessed by an MTT assay. The results shown are the mean and standard deviation of three independent experiments. *Significantly (p < 0.05) different from cells without DHEA or pentachlorophenol treatment. (B) The levels of human SULT2A1 were assayed using Western blot. Cytosolic proteins (10–40 μg/well) were loaded. β-Actin was used as a loading control. (C) The levels of 12-sulfoxynevirapine in the culture medium of TK6/SULT2A1 cells incubated with 90 μM 12-hydroxynevirapine, in the presence or absence of the SULT2A1 substrates DHEA (5 μM) or pentachlorophenol (10 μM) for 48 h. Data are the mean and standard deviation of three independent experiments. *Significantly (p < 0.05) different from cells without DHEA or pentachlorophenol treatment.

4. Discussion

Although sulfonation is generally considered as a detoxification pathway by producing more water-soluble and often less toxic metabolites, the sulfonation of benzylic hydroxyl groups has been associated with the formation of reactive electrophiles. This has been demonstrated with the sulfate esters of 7-hydroxymethyl-12-methylbenz[a] anthracene, 6-hydroxymethybenzo[a]pyrene, 9-hydroxymethyl-10-methylanthracene, 1-hydroxymethylpyrene, and 10-hydroxysafrole, which have been shown to bind covalently to DNA and proteins to produce both toxic and carcinogenic responses [26].

Previous studies have reported the presence of 12-sulfoxynevirapine in the urine and bile from rats administered nevirapine [8,15]. This is the first study to characterize the sulfonation of 12-hydroxynevirapine by human SULT isoforms. Several human SULTs were shown to exhibit significant levels of sulfonation activity towards 12-hydroxynevirapine. Whereas the SULT with the highest Vmax/Km ratio and therefore the highest overall catalytic activity towards 12-hydroxynevirapine was SULT2A1, other SULTs, including SULT1A1, SULT1A2, SULT1B1, SULT1C4, and SULT1E1, also catalyzed the sulfonation of this metabolite. It is noteworthy that there was no formation of 12-sulfoxynevirapine in the reactions using mouse liver cytosols, which contrasts with rat liver cytosols (Table 1).

In the current study, 12-sulfoxynevirapine was synthesized and structurally characterized. The reaction of 12-sulfoxynevirapine with deoxynucleosides occurred very slowly (at 37 °C for 15 days) with < 0.01% of 12-sulfoxynevirapine being bound to deoxynucleosides, indicating that deoxynucleosides are not the major targets for adduct formation. Although 12-sulfoxynevirapine was found to be much less reactive than 12-mesyloxynevirapine, a surrogate for 12-sulfoxynevirapine used in the previous studies [16,19], these deoxynucleoside adducts could plausibly be formed in vivo via sulfonation of 12-hydroxynevirapine. More importantly, some of these adducts, such as 12-(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, and 12-(guanin-N7-yl)nevirapine, may be potentially mutagenic. Additional studies are necessary to determine if these adducts are formed in vivo.

More recently, nevirapine amino acid adducts have been characterized using 12-mesyloxynevirapine as a surrogate for 12-sulfoxynevirapine [17,18]. In this study, we demonstrated that 12-sulfoxynevirapine reacted with cysteine and glutathione to form adducts, although to a lesser extent than observed with 12-mesyloxynevirapine. The structures of the purified cysteine and glutathione adducts resulting from the reaction with 12-sulfoxynevirapine were consistent with the previous reports [17,18].

As indicated above, SULT1A1 has sulfonation activity towards 12-hydroxynevirapine. In a previous study, very low protein levels of SULT1A1 and SULT2B1 were detected in HEK293 and HEK293/SULT vector cells [24]. The background SULT1A1sulfonation activity towards 12-hydroxynevirapine in parent HEK293 cells could be confounder, which may explain why 12-hydroxynevirapine treatment caused a similar cytotoxicity among HEK293, HEK293/SULT vector, and HEK293/SULT2A1 cells [27].

The human TK6 lymphoblastoid cell line has several desirable properties, such as human origin, a high efficiency of cell proliferation, and the expression of wild-type p53 protein [28]. TK6 cells readily grow in culture and have been widely used in genotoxic and cytotoxic research. Furthermore, the lack of biotransformation processes in TK6 cells [29] makes them a good model to overexpress metabolic enzymes of interest. Our study confirmed the lack of sulfonation activity towards both triclosan and 12-hydroxynevirapine in TK6 cells. Given that SULT2A1 has the highest overall catalytic activity towards 12-hydroxynevirapine, a TK6/SULT2A1 cell line overexpressing SULT2A1 was constructed to address the potential involvement of the sulfonation pathway in the cytotoxicity induced by 12-hydroxynevirapine.

Nevirapine inhibited the cell growth of both human hepatoma HepG2 cells and immortalized normal liver THLE2 cells at a concentration of approximately 1.0 mM following a 48 h exposure [30]. In the present study, a similar effect was observed in TK6 cells and SULT2A1-overexpressing cell line generated from TK6 cells, but at a much lower concentration of nevirapine (approximately 130 μM). Further, 12-hydroxynevirapine was found to be more cytotoxic to all three TK6 cell lines than nevirapine, which is consistent with the previous findings in rats that lower doses of 12-hydroxynevirapine induced the same degree of skin rash as the treatment with nevirapine itself and that the incidence of skin rash was correlated with the blood levels of 12-hydroxynevirapine [15]. These results indicate that 12-hydroxynevirapine is a key metabolite involved in the toxicity associated with nevirapine.

Subsequent sulfonation of 12-hydroxynevirapine led to less cytotoxicity in TK6/SULT2A1 cells compared to TK6 and TK6/SULT vector cells. Co-treatment of TK6/SULT2A1 cells with 12-hydroxynevirapine and DHEA, a competitive SULT2A1 substrate [31,32], slightly enhanced the cytotoxicity induced by 12-hydroxynevirapine in TK6/SULT2A1 cells and decreased the levels of 12-sulfoxynevirapine. Similar incubations were conducted with 12-hydroxynevirapine and pentachlorophenol, which is also a substrate for SULT2A1 [31], and, unexpectedly, there was a decrease in cytotoxicity and an increase in the levels of 12-sulfoxynevirapine. Although the reasons for the anomalous behavior with pentachlorophenol are currently unknown, the results obtained with both DHEA and pentachlorophenol clearly support the interpretation that sulfonation of 12-hydroxynevirapine attenuates its cytotoxicity.

In conclusion, we have demonstrated that 12-sulfoxynevirapine can form adducts with deoxynucleosides and amino acids, with the reactivity being very low with deoxynucleosides. Several human SULTs catalyzed the sulfonation of 12-hydroxynevirapine and sulfonation of 12-hydroxynevirapine decreased its cytotoxicity in human TK6 lymphoblastoid cells.

Acknowledgements

JLF conceived and designed the study, synthesized the 12-sulfoxynevirapine, conducted its reaction with deoxynucleosides and amino acids, analyzed the sulfonation of 12-hydroxynevirapine by human SULT isoforms, generated the SULT2A1 overexpressing TK6 cell line, determined the cytotoxicity of nevirapine and 12-hydroxynevirapine in TK6/SULT2A1 cells, performed the statistical analysis, and drafted the manuscript. LL characterized the adducts of 12-sulfoxynevirapine with deoxynucleosides and amino acids and helped to draft the manuscript. PC characterized the 12-sulfoxynevirapine and helped to draft the manuscript. GGC helped to characterize the 12-sulfoxynevirapine and its adducts. FAB participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript. The authors declare that they have no conflict of interest. This study was supported through an interagency agreement between the National Center for Toxicological Research, U.S. Food and Drug Administration and the National Toxicology Program, National Institute of Environmental Health Sciences (FDA IAG: 224-07-0007; NIH Y1ES1027).

Footnotes

☆

The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.

References

  • [1].Clotet B, Once-daily dosing of nevirapine in HAART, J. Antimicrob. Chemother 61 (1) (2008) 13–16. [DOI] [PubMed] [Google Scholar]
  • [2].Lockman S, Shapiro RL, Smeaton LM, Wester C, Thior I, Stevens L, Chand F, Makhema J, Moffat C, Asmelash A, Ndase P, Arimi P, van Widenfelt E, Mazhani L, Novitsky V, Lagakos S, Essex M, Response to antiretroviral therapy after a single, peripartum dose of nevirapine, N. Engl. J. Med 356 (2) (2007) 135–147. [DOI] [PubMed] [Google Scholar]
  • [3].Taiwo BO, Nevirapine toxicity, Int. J. STD AIDS 17 (6) (2006) 364–369. [DOI] [PubMed] [Google Scholar]
  • [4].PDR, Viramune (Nevirapine). Physicians’ Desk Reference, Physicians’ Desk Reference, Medical Economics Co., Montvale, NJ, 2009, pp. 873–881. [Google Scholar]
  • [5].Manosuthi W, Thongyen S, Chumpathat N, Muangchana K, Sungkanuparph S, Incidence and risk factors of rash associated with efavirenz in HIV-infected patients with preceding nevirapine-associated rash, HIV Med 7 (6) (2006) 378–382. [DOI] [PubMed] [Google Scholar]
  • [6].Calmy A, Hirschel B, Cooper DA, Carr A, A new era of antiretroviral drug toxicity, Antivir. Ther 14 (2) (2009) 165–179. [DOI] [PubMed] [Google Scholar]
  • [7].Marinho AT, Rodrigues PM, Caixas U, Antunes AMM, Branco T, Harjivan SG, Marques MM, Monteiro EC, Pereira SA, Differences in nevirapine biotransformation as a factor for its sex-dependent dimorphic profile of adverse drug reactions, J. Antimicrob. Chemother 69 (2) (2014) 476–482. [DOI] [PubMed] [Google Scholar]
  • [8].Srivastava A, Lian L-Y, Maggs JL, Chaponda M, Pirmohamed M, Williams DP, Park BK, Quantifying the metabolic activation of nevirapine in patients by integrated applications of NMR and mass spectrometries, Drug Metab. Dispos 38 (1) (2010) 122–132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Fan-Havard P, Liu Z, Chou M, Ling Y, Barrail-Tran A, Haas DW, Taburet A-M, ANRS12154 Study Group, Pharmacokinetics of phase I nevirapine metabolites following a single dose and at steady state, Antimicrob. Agents Chemother 57 (5) (2013) 2154–2160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Ren C, Fan-Havard P, Schlabritz-Loutsevitch N, Ling Y, Chan KK, Liu Z, A sensitive and specific liquid chromatography/tandem mass spectrometry method for quantification of nevirapine and its five metabolites and their pharmacokinetics in baboons, Biomed. Chromatogr 24 (7) (2010) 717–726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Liu Z, Fan-Havard P, Xie Z, Ren C, Chan KK, A liquid chromatography/atmospheric pressure ionization tandem mass spectrometry quantitation method for nevirapine and its two oxidative metabolites, 2-hydroxynevirapine and nevirapine 4-carboxylic acid, and pharmacokinetics in baboons, Rapid Commun. Mass Spectrom 21 (16) (2007) 2734–2742. [DOI] [PubMed] [Google Scholar]
  • [12].Riska P, Lamson M, MacGregor T, Sabo J, Hattox S, Pav J, Keirns J, Disposition and biotransformation of the antiretroviral drug nevirapine in humans, Drug Metab. Dispos 27 (8) (1999) 895–901. [PubMed] [Google Scholar]
  • [13].Riska PS, Joseph DP, Dinallo RM, Davidson WC, Keirns JJ, Hattox SE, Biotransformation of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs, monkeys, and chimpanzees, Drug Metab. Dispos 27 (12) (1999) 1434–1447. [PubMed] [Google Scholar]
  • [14].Erickson DA, Mather G, Trager WF, Levy RH, Keirns JJ, Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic cytochromes P-450, Drug Metab. Dispos 27 (12) (1999) 1488–1495. [PubMed] [Google Scholar]
  • [15].Chen J, Mannargudi BM, Xu L, Uetrecht J, Demonstration of the metabolic pathway responsible for nevirapine-induced skin rash, Chem. Res. Toxicol 21 (9) (2008) 1862–1870. [DOI] [PubMed] [Google Scholar]
  • [16].Antunes AMM, Duarte MP, Santos PP, Gamboa da Costa G, Heinze TM, Beland FA, Marques MM, Synthesis and characterization of DNA adducts from the HIV reverse transcriptase inhibitor nevirapine, Chem. Res. Toxicol 21 (7) (2008) 1443–1456. [DOI] [PubMed] [Google Scholar]
  • [17].Antunes AMM, Godinho ALA, Martins IL, Justino GC, Beland FA, Marques MM, Amino acid adduct formation by the nevirapine metabolite, 12-hydroxynevirapine – a possible factor in nevirapine toxicity, Chem. Res. Toxicol 23 (5) (2010) 888–899. [DOI] [PubMed] [Google Scholar]
  • [18].Antunes AMM, Godinho ALA, Martins IL, Oliveira MC, Gomes RA, Coelho AV, Beland FA, Marques MM, Protein adducts as prospective biomarkers of nevirapine toxicity, Chem. Res. Toxicol 23 (11) (2010) 1714–1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Antunes AMM, Wolf B, Oliveira MC, Beland FA, Marques MM, 2’-Deoxythymidine adducts from the anti-HIV drug nevirapine, Molecules 18 (5) (2013) 4955–4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Caixas U, Antunes AMM, Marinho AT, Godinho ALA, Grilo NM, Marques MM, Oliveira MC, Branco T, Monteiro EC, Pereira SA, Evidence for nevirapine bioactivation in man: searching for the first step in the mechanism of nevirapine toxicity, Toxicology 301 (1–3) (2012) 33–39. [DOI] [PubMed] [Google Scholar]
  • [21].Meng X, Howarth A, Earnshaw CJ, Jenkins RE, French NS, Back DJ, Naisbitt DJ, Park BK, Detection of drug bioactivation in vivo: mechanism of nevirapine-albumin conjugate formation in patients, Chem. Res. Toxicol 26 (4) (2013) 575–583. [DOI] [PubMed] [Google Scholar]
  • [22].Gamage N, Barnett A, Hempel N, Duggleby RG, Windmill KF, Martin JL, McManus ME, Human sulfotransferases and their role in chemical metabolism, Toxicol. Sci 90 (1) (2006) 5–22. [DOI] [PubMed] [Google Scholar]
  • [23].Glatt H, Sulfotransferases in the bioactivation of xenobiotics, Chem. Biol. Interact 129 (1–2) (2000) 141–170. [DOI] [PubMed] [Google Scholar]
  • [24].Fang J-L, Wu Y, Gamboa da Costa G, Chen S, Chitranshi P, Beland FA, Human sulfotransferases enhance the cytotoxicity of tolvaptan, Toxicol. Sci 150 (1) (2016) 27–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Boysen G, Pachkowski BF, Nakamura J, Swenberg JA, The formation and biological significance of N7-guanine adducts, Mutat. Res 678 (2) (2009) 76–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Michejda CJ, Kroeger Koepke MB, Carcinogen activation by sulfate conjugate formation, Adv. Pharmacol 27 (1994) 331–363. [DOI] [PubMed] [Google Scholar]
  • [27].Fang J-L, Wu Y, Loukotková L, Chitranshi P, Gamboa da Costa G, Beland FA, Cytotoxicity of 12-sulfoxynevirapine, The Toxicologist SOT 2017 Annual Meeting (Abstract 2234) (2017) 292. [Google Scholar]
  • [28].Platel A, Nesslany F, Gervais V, Claude N, Marzin D, Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: determination of No-Observed-Genotoxic-Effect-Levels, Mutat. Res 726 (2) (2011) 151–159. [DOI] [PubMed] [Google Scholar]
  • [29].Barile FA, Principles of Toxicology Testing/Chapter 17 Mutagenicity and Carcinogenicity Testing in Vitro, CRC Press Taylor & Francis Group, New York, USA, 2013, pp. 229–255. [Google Scholar]
  • [30].Fang J-L, Beland FA, Differential responses of human hepatocytes to the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine, J. Toxicol. Sci 38 (5) (2013) 741–752. [DOI] [PubMed] [Google Scholar]
  • [31].Gulcan HO, Liu Y, Duffel MW, Pentachlorophenol and other chlorinated phenols are substrates for human hydroxysteroid sulfotransferase hSULT2A1, Chem. Res. Toxicol 21 (8) (2008) 1503–1508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Wang L-Q, James MO, Inhibition of sulfotransferases by xenobiotics, Curr. Drug Metab 7 (1) (2006) 83–104. [DOI] [PubMed] [Google Scholar]

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