M. Backlund - Academia.edu (original) (raw)

Papers by M. Backlund

Transcriptional and post-translational regulation of CYP1A1 by primaquine

The Journal of pharmacology and experimental therapeutics, 2001

Regulation of the CYP1A1 gene has been shown to involve the aryl hydrocarbon receptor and the CYP... more Regulation of the CYP1A1 gene has been shown to involve the aryl hydrocarbon receptor and the CYP1A1 gene expression is induced by AhR ligands. Primaquine is an antimalarial agent that does not exhibit the structural properties of a classical AhR ligand. We have evaluated the mechanisms by which this compound induces CYP1A1 expression using rat hepatoma H4IIE cells and V79 cells stably expressing CYP1A1. In H4IIE cells, primaquine caused a time- and dose-dependent increase of CYP1A1 mRNA and protein expression. The transcriptional activation of the CYP1A1 gene by primaquine was strictly XRE-dependent, as shown by transfection of different CYP1A1 pGL3 reporter constructs in H4IIE cells, and the involvement of the AhR was shown by activation of a Gal4-AhR hybrid protein by primaquine in transfected cells. Furthermore, primaquine caused transformation of the cytosolic AhR to a DNA-binding form, in vitro, suggesting that primaquine directly activates the receptor complex. In addition to...

Cytochrome P4502D4 in the brain: specific neuronal regulation by clozapine and toluene

Molecular pharmacology, 1996

Twenty-four hr after a single dose of the neuroleptic drug clozapine, cytochrome P4502D4 (P4502D4... more Twenty-four hr after a single dose of the neuroleptic drug clozapine, cytochrome P4502D4 (P4502D4) immunoreactivity, which was barely detectable in the brains of untreated rats, was clearly evident in neurons of the substantia nigra pars compacta, ventral tegmental area, granular neurons of the olfactory bulb, and Purkinje and granular neurons of the cerebellum. Induction was maintained with daily administration for 3 weeks. The mRNA for P4502D4 was detected by Northern blotting and localized by in situ hybridization in neurons throughout the brain and in the Bergman glia in the cerebellum. There were no detectable changes in the distribution or quantity of P4502D4 mRNA after treatment with clozapine. The overall P450 content of the brain increased with daily administration to a approximately 7-fold induction by 3 weeks of clozapine treatment. No induction of 2D4 was observed with the dopamine D2 receptor blockers haloperidol, chlorpromazine, and sulpiride or with the serotonin rece...

Functional polymorphism in the alcohol dehydrogenase 3 (ADH3) promoter

Pharmacogenetics, 2001

The ADH3 gene encodes alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydroge... more The ADH3 gene encodes alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase, the ancestral and most conserved form of alcohol dehydrogenase. ADH3 is expressed in all tissues examined and the enzyme is essential for formaldehyde scavenging. We have screened the promoter region including exon 1 and exons 5, 6 and 7 of the ADH3 gene for allelic variants. Using 80 samples of genomic DNA from Swedes as template, the various parts of the gene were PCR amplified and subsequently analyzed on single strand conformation polymorphism (SSCP) gels. No abnormal migration patterns could be detected by SSCP analysis of exons 5, 6 and 7 while for the promoter region, a large number of the samples displayed differences in SSCP gel migration patterns. Cloning and sequence analysis revealed four possible base pair exchanges in the promoter region. Two transitions were found at position -197 and -196, GG --> AA, one at position -79, G --> A and finally, close to the trans...

Structural and mechanistic aspects of transcriptional induction of cytochrome P450 1A1 by benzimidazole derivatives in rat hepatoma H4IIE cells

European Journal of Biochemistry, 1999

The effect of several structurally different benzimidazole compounds on CYP1A1 expression at the ... more The effect of several structurally different benzimidazole compounds on CYP1A1 expression at the transcriptional, mRNA and protein levels was investigated in the rat hepatoma H4IIE cell line. Omeprazole, thiabendazole, carbendazim, 2-mercaptobenzimidazole and 2-mercapto-5-methoxybenzimidazole caused a dose-dependent increase in CYP1A1 protein levels that reached maximum effect at 250 microm, as measured by Western blot. In addition, hydroxyomeprazole, 2-aminobenzimidazole and 2-mercapto-5-nitro-benzimidazole caused a notable increase in CYP1A1 protein expression, whereas 5-O-desmethylomeprazole, 2-hydroxybenzimidazole, 2-benzimidazole propionic acid and 5-benzimidazole carboxylic acid were ineffective. Thus, benzimidazole substituted with a thiol or an amino group in the 2-position were active inducers. Northern blot analysis confirmed an extensive increase of CYP1A1 mRNA induced by omeprazole and 2-mercapto-5-methoxybenzimidazole which was 32% and 49% of maximal induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) respectively, whereas thiabendazole and carbendazim showed approximately 15% increase as compared to TCDD. Transient transfection of H4IIE cells, with a XRE-pGL3 reporter gene construct revealed a 2.3-4.3-fold induction by carbendazim, thiabendazole, and 2-mercapto-5-methoxybenzimidazole as compared to a 3.3- and 23-fold induction by omeprazole and TCDD, respectively. Thus, these data indicate that the benzimidazoles utilize the aryl hydrocarbon receptor-arnt-XRE-mediated signal-transduction pathway for induction of the CYP1A1 gene.

Chemico-Biological Interactions, 2010

UVB induces the expression of genes controlled by the aryl hydrocarbon receptor (AhR), a transcri... more UVB induces the expression of genes controlled by the aryl hydrocarbon receptor (AhR), a transcription factor that has been implicated in the UV stress response. In this study, we used the human hepatoma cell line HepG2 to investigate in more detail the effects of UVB irradiation on AhR activation and induction of cytochrome P450 1A1 (CYP1A1), a highly AhR-responsive gene. The CYP1A1 enzyme efficiently degrades 6-formylindolo[3,2-b]carbazole (FICZ), a high affinity ligand and suggested endogenous activator of the AhR. We show that physiologically relevant doses of UVB suppress CYP1A1 gene expression immediately after irradiation, but induce its expression later in an AhR-dependent manner. The initial repression phase of CYP1A1 transcription was mediated by another UVB-inducible transcription factor, the nuclear factor B (NFB). Crosstalk between AhR and NFB signaling has earlier been implicated to control CYP1A1 expression following stimulation by xenobiotics and cytokines. Now, our findings clearly indicate a role of NFB also in UVB-dependent AhR signaling. We also observed that UVB reduced the catalytic activity of the CYP1A1 enzyme. Thereby, UVB attenuated the clearance of FICZ, which led to prolonged AhR activation. We further noted that repeated irradiation with UVB or H 2 O 2 treatment shifted the cells into a refractory state in which AhR signaling could not be efficiently activated by UVB or H 2 O 2 , but by ligands. Together, our results suggest that the NFB-mediated initial suppression of CYP1A1 as well as the unresponsiveness of AhR signaling to repeated irradiation may be part of a protective cellular UV stress response.

Regulation of aryl hydrocarbon receptor signal transduction by protein tyrosine kinases

Cellular Signalling, 2005

The involvement of protein tyrosine kinases (PTKs) in aryl hydrocarbon receptor (AhR)-mediated si... more The involvement of protein tyrosine kinases (PTKs) in aryl hydrocarbon receptor (AhR)-mediated signalling by omeprazole and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in hepatoma cells. Both omeprazole- and TCDD-dependent AhR signalling was attenuated by inhibition of c-src kinase, either by using pyrazolopyrimidine 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4 ]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitors or by expression of dominant-negative c-src. These results indicate that the overall AhR function is modulated by c-src kinase activity. In contrast, a selective inhibition of omeprazole-mediated AhR signalling was revealed by tyrosine kinase inhibitors, tyrphostins AG17 and AG879. Furthermore, omeprazole-dependent AhR activation was abolished by mutation of Tyr320 to Phe, suggesting that this residue is a putative phosphorylation site. TCDD-dependent AhR signalling was neither affected by tyrphostins nor by this mutation. Our results are consistent with activation of the AhR by omeprazole in a ligand-independent manner, via a signal transduction pathway that involves protein tyrosine kinases, and are different from the mechanism exerted by high-affinity ligands.

Biotechnology and Bioengineering, 1993

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipa... more The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water-in-oil microemulsion (AOThsooctane). By using n-propanol a s the alcohol, an optimal wo ([H201/[AOTl ratio) of 12 was found for the synthesis of n-propylibuprofenate at room temperature. The lipase showed high preference for the S(+)-enantiomer of ibuprofen, which was esterified to the corresponding S(+)-ibuprofen ester. The R(-)-ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S-ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n-alcohols with different chain lengths (3-12) were compared, and it appeared that short-(propanol and butanol) and long-chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed t o the corresponding S-ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S-ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S-ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due t o interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems.

Biochemical and Biophysical Research Communications, 2004

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and ... more A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.

Biocatalysis and Biotransformation, 1992

Molecular Pharmacology, 2004

The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is ... more The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is responsible for the regulation of several response genes, of which the best characterized is the CYP1A1 gene. The present study was undertaken to elucidate the mechanism of activation of the AhR by omeprazole (OME), 2-mercapto-5-methoxybenzimidazole (MMB), and primaquine (PRQ), compounds that have previously been reported to induce CYP1A1 expression but that are not typical AhR ligands. All compounds caused a significant increase in luciferase activity in rat H4IIE and human HepG2 hepatoma cells transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. Furthermore, MMB and PRQ, but not OME, were capable of transforming cytosolic AhR to a DNA-binding form and displacing AhR-bound [ 3 H]TCDD in rat hepatic cytosol in vitro. By performing site-directed mutagenesis of residues in the ligand-binding domain of the Gal4-AhR, a construct containing a Y320F substitution was found to be resistant to activation by OME, MMB, and PRQ, but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparable affinities of [ 3 H]TCDD-binding to the wild-type and the Y320F mutant Gal4-proteins, expressed in human embryonic kidney 293 cells, were obtained in the ligand-binding assay. In contrast, the competition of receptor-bound [ 3 H]TCDD by PRQ was absent from Gal4-Y320F but not from Gal4-AhR cell extracts. The results of this study confirm that MMB and PRQ are low-affinity ligands for the AhR and suggest that high-and low-affinity ligands interact with different residues of the AhR ligand-binding pocket. In addition, the data presented here indicate that Tyr 320 plays an important role in AhR activation. The aryl hydrocarbon receptor (AhR) is a member of the growing family of basic helix-loop-helix Per-Arnt-Sim (PAS) transcription factors whose members play key roles in development, adaptation to hypoxia, control of circadian rhythmicity, and metabolism of xenobiotic compounds (Gu et al., 2000). Mechanistically, the AhR functions as a ligand-activated transcription factor that is responsible for the transcriptional activation of several AhR-responsive genes (see reviews by Hankinson, 1995; Whitlock, 1999). A variety of environmental pollutants (e.g., polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons) are high-affinity ligands for the AhR. These ligands, including the prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are characterized by being planar, aromatic, and hydrophobic molecules, fitting into a ligand-binding pocket with a maximal dimension of 14 ϫ 12 ϫ 5 Å. In addition, electronic and thermodynamic properties of the ligand have been shown to be critical for favorable interactions between the ligand and the receptor (Gillner et al., 1993; Kafafi et al., 1993; Waller and McKinney, 1995). Recent identification and characterization of novel AhR ligands or AhR activators (reviewed by Denison and Nagy, 2003), which have physicochemical and structural properties that deviate significantly from these typical AhR ligands, challenge the currently defined ligand-binding model. The structural diversity of these atypical AhR ligands/activators is clearly evident by comparison of the molecular structures of,

Toxicological Sciences, 2006

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as a... more Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as additives in various consumer products. Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like properties. We studied the ability of environmentally relevant PBDEs and the "planar" congener BDE-77 to bind and/or activate the AhR in stably transfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-Odee-thylation (EROD) was used as a marker for CYP1A1 activity. Dose-and bromination-specific inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured by their ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1 activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings were observed after exposing stably transfected human hepatoma (xenobiotic response element [XRE]-HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects, confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only due to direct interaction at receptor level but also at DNA-binding level. This antagonism was confirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation and dioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, the transactivation function of the BDE-99-activated AhR seems to be very weak. These combined results suggest that PBDEs do bind but not activate the AhR-AhR nuclear translocator protein-XRE complex.