Subscriber access provided by NEW YORK UNIV
Article
Human sulfotransferase 1A1 dependent mutagenicity of 12-hydroxy-Nevirapine: the missing link? Michel Kranendonk, Mónica Alves, Pedro Antunes, and José Rueff Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx5003113 • Publication Date (Web): 02 Oct 2014 Downloaded from http://pubs.acs.org on October 11, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Chemical Research in Toxicology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Chemical Research in Toxicology
Human sulfotransferase 1A1 dependent mutagenicity of 12hydroxy-nevirapine: the missing link? Michel Kranendonk*, Mónica Alves†, Pedro Antunes and José Rueff Department of Genetics/CIGMH, NOVA Medical School/Faculdade Ciências Médicas, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisbon, PORTUGAL KEYWORDS: nevirapine, bioactivation, reactive metabolites, mutagenicity, sulfotransferase, cytochrome P450, adverse drug reaction, carcinogenicity
ABSTRACT Nevirapine (NVP) is a frequently used anti-HIV drug. Despite its efficacy, NVP has been associated with serious skin and liver injuries in exposed patients and with increased incidences of hepato-neoplasias in rodents. Current evidence supports the involvement of reactive metabolites in the skin- and liver-toxicities, formed by cytochrome P450 mediated oxidations and/or subsequent Phase II sulfonation. However, to date standard in vitro genotoxicity tests have provided no evidence that NVP is either mutagenic or clastogenic. The human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-NVP, one of the major metabolites of NVP, is demonstrated here.
Nevirapine (NVP) is a widely prescribed non-nucleoside inhibitor of HIV-1 reverse transcriptase (NNRTI), the first one approved by the FDA in 1996. It is effective when used as part of combination therapy to treat HIV-1-infected individuals and as monotherapy for prevention of mother-to-child HIV-1 transmission.1 Despite its therapeutic efficacy, in particular against vertical HIV-transmission, severe skin and liver-injury has been reported, although individual susceptibilities for NVP adverse effects differ among patients.2, 3 NVP has also been associated with increased incidences of hepato-neoplasias in rodents.4 Additionally, epidemiological data suggest that NNRTI use in general, is a risk factor for non-AIDS defining cancers in HIV-positive patients.5
Figure 1. Chemical structure of NVP
Current evidence indicates generation of reactive NVP metabolites as the culprit of its associated liver and skin toxicity. NVP is metabolized at first instance through oxidative metabolism mediated by cytochrome P450s, by hydroxylation at the C3-, C8- (both mainly by CYP2B6), C2- or C12-position (latter two mainly by CYP3A4) (Figure 1).6 In particular oxidative metabolism at the C12 position has been implicated in NVP induced toxicity. The reactive C4-C12 quinone methide was previously suggested as the reactive species, formed ei-
ther directly by cytochrome P450 mediated dehydrogenation or by C12-hydroxylation, sulfonation and subsequent sulfate loss.7 Protein adducts could be detected using in vitro approaches for 12-sulfoxy-NVP in serum albumin and glutathione S-transferase π, and for the synthetic surrogate 12mesyloxy NVP with hemoglobin.8-10 Several of these protein adducts were detected in blood of patients receiving NVP, leaving strong indications of NVP haptenation and causal role in these associated adverse drug reactions.8, 11 Currently, evidence suggests the involvement of the NVP quinone methide in the induction of hepato-toxicity while the 12-sulfoxy-NVP metabolite is responsible for skin rash, with a possible prominent role for sulfotransferase 1A1 (SULT1A1).12-14 The synthetic surrogate for 12-sulfoxy-NVP, 12-mesyloxyNVP has been demonstrated to be reactive with DNA.15, 16 The induction of hepato-adenomas and carcinomas in rodents as well as the increased risk for non-AIDS defining cancers with long-term NNRTI use raises concerns on possible genotoxicity of NVP.4, 5 Still, standard in vitro mutagenicity tests have provided no evidence that NVP is either mutagenic or clastogenic.4 These standard assays use exogenous liver derived metabolic systems and reactive sulfoxy ester intermediates are thus generated externally, demonstrating often limited penetration capacity of the indicator cells due to their increased hydrophilicity.17 As such, sulfotransferase mediated bioactivation is not detected in standard mutagenicity assays, a possible explanation for the discrepancy between the in vitro and in vivo outcomes of NVP genotoxity testing.17 The objective of this current study was to verify if 12OH-NVP is mutagenic in a SULT dependent manner. Several human sulfotransferase competent Ames tester strains have been developed previously by the group of Dr. Hansruedi Glatt (Germany), including those expressing human SULT1A1.17-21 Although these have been demonstrated to be quite successful in detecting mutagenicity of specific sulfoxy-
ACS Paragon Plus Environment
Chemical Research in Toxicology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 6
esters, some drawbacks were noted by these authors (see Supporting Information).20 To overcome these issues, new human SULT1A1 expressing Ames tester bacteria were developed, namely MA98_SULT1A1 and MA100_SULT1A1, both demonstrating controllable and stable SULT1A1 expression (see Supporting Information).
Figure 3. PCP inhibition plot of 1-HMP (8.6 pmol) induced mutagenicity in strain MA98_SULT1A1
The metabolite 12OH-NVP was tested with the two new SULT1A1 strains, first applying a logarithmic dose-gradient (Figure 4A), testing over 4 orders of magnitude in dose-levels.
Figure 2. Dose-response curves of 1-HMP (A) with strains MA98_SULT1A1 and TA98 and of FFA (B) with strains MA100_SULT1A1 plus TA100
The SULT1A1 dependent mutagens FFA and 1-HMP were tested with these new human SULT1A1 competent Ames strains to verify their sensitivity in the detection of SULT1A1dependent mutagens (Figure 2A and 2B).18, 21 FFA induced 198 ± 19 His+ revertant colonies per nmol. This was 81 ± 10 revertant per pmol for 1-HMP with strain MA98_SULT1A1. These mutagenic activities for FFA and 1-HMP are respectively 22- and 6-fold higher as those reported previously for these compounds with SULT1A1 expressing Ames strains.18, 21 Both compounds did not demonstrate any mutagenicity for the same dose-gradients with the comparable SULT1A1-empty strains TA98 and TA100 (Figure 2A and B). The new SULT1A1 tester strains were further characterize in what concerns their SULT1A1 bioactivation capacity, using pentachlorophenol (PCP), a potent inhibitor of SULT1A1.22 The PCP-inhibition study with strain MA98_SULT1A1 (Figure 3) demonstrated a very efficient inhibition of 1-HMP induced mutagenicity, demonstrating an IC50 value of 0.17 µM (r2=0.974).
Figure 4. Dose-response curves of 12OH-NVP with (A) logarithmic or (B) linear dose gradient (dose-response curve of MA98_SULT1A1 in (B) represents the average of 4 independent experiments).
MA98_SULT1A1 demonstrated a positive response, which is not the case for MA100_SULT1A1, indicating a preference for inducing frameshift mutations. Of note, both strains which are derivatives of strain TA98 and TA100, contain in their DNA G/C stretches, part of their L-His-reversion targets,
ACS Paragon Plus Environment
Page 3 of 6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Chemical Research in Toxicology
corroborating the reactivity of the synthetic 12-mesyloxy-NVP with deoxyguanosine and deoxycytidine.16, 23 When tested with a linear dose-gradient 12OH-NVP demonstrated a mutagenic activity of 34 ± 1 revertant colonies per µmol, with strain MA98_SULT1A1 (Figure 4B). 12OH-NVP demonstrated no mutagenicity with the comparable SULT1A1-empty strain TA98, for the same dose-levels (Figure 4B), indicating the bioactivation role of human SULT1A1 in the mutagenicity of 12OH-NVP. The SULT1A1 inhibitor PCP was used to further confirm the SULT1A1-bioactivation role in 12OH-NVP mutagenicity.22 When applying 1.8 µM PCP (approx. 10-fold of the IC50 concentration, vide supra), a complete abolishment of 12OH-NVP’s mutagenicity was observed (p
