Abstract
Background/Aim: Phenolic compounds, particularly dihydroxybiphenyl-related compounds, possess efficient anti-oxidative and anti-inflammatory activity. We investigated the anti-inflammatory activity of 2,2’-dihydroxy-5,5’-dimethylbiphenol (p-cresol dimer), 2,2’-dihydroxy-5,5’-dimethoxybiphenol (pHA dimer), p-cresol, p-hydroxyanisole (pHA) and 2-t-butyl-4-hydroxyanisole (BHA). Materials and Methods: The cytotoxicity of the investigated compounds against RAW264.7 cells was determined using a cell counting kit (CCK-8). Their inhibitory effects on cyclooxygenase-2 (Cox2) mRNA expression stimulated by lipopolysaccharide (LPS) were determined using northern blot analysis, and their inhibition of LPS-stimulated nuclear factor-kappa B (Nf-κb) activation was evaluated using enzyme-linked immunosorbent assay-like microwell colorimetric transcription factor activity assay. The molecular orbital energy was calculated on the basis of density function theory BLYP/6-31G*. Results: The cytotoxicity of the compounds declined in the order pHA dimer > p-cresol dimer > BHA > p-cresol > pHA. The inhibitory effect on Cox2 expression and Nf-κb activation was enhanced by p-cresol dimer and pHA dimer, particularly the former, suggesting potent anti-inflammatory activity, whereas p-cresol and pHA showed weak activity, and BHA no activity. Both p-cresol dimer and pHA dimer were highly electronegative, as determined by quantum chemical calculations. Conclusion: Dimerization of p-cresol and pHA enhances their anti-inflammatory activity. p-Cresol dimer and pHA dimer, particularly the former, are potent anti-inflammatory agents.
- p-Cresol dimer
- p-hydroxy anisole dimer
- antioxidative activity
- RAW264.7 cells
- cytotoxicity
- Cox2
- Nf-κb
- quantum molecular properties
Phenolic compounds possess efficient antioxidative activity and are potentially useful bioactive agents. p-Cresol (4-methylphenol; Figure 1) is a natural product present in many foods, crude oil, and coal tar, and also detectable in animal and human urine. In addition to its industrial uses, p-cresol is also used as an antiseptic and disinfectant because of its bactericidal and fungicidal properties, but there are still insufficient data to support its safety for use in cosmetics (1). p-Hydroxyanisole (Figure 1) is also an acceptable anti-oxidant, but is known to be carcinogenic, being structurally similar to the known forestomach carcinogen, BHA (2-t-butyl-4-hydroxyanisole, Figure 1) (2, 3). We previously investigated the anti-oxidative activity, cytotoxicity and anti-inflammatory activity of BHA and 3,3’-di-t-butyl-5,5’-dimethoxy-1,1’-biphenyl-2,2’-diol (bis-BHA), a BHA orthodimer, and demonstrated that, unlike BHA, it exerted potent anti-oxidative and anti-inflammatory activity, although the molecular mechanism responsible for the biological activities of these compounds was not completely clarified (4). We also previously synthesized 2,2’-dihydroxy-5,5’-dimethylbiphenol, (p-cresol dimer) and 2,2’-dihydroxy-5,5’-dimethoxybiphenol (pHA dimer) by ortho-ortho coupling reactions with the parent monomers, p-cresol and pHA, respectively, and investigated their anti-oxidative activity and cytotoxicity toward the murine macrophage-like cell line, RAW264.7, demonstrating that like BHA, p-cresol dimer and pHA dimer possessed effective anti-oxidative activity and also acceptable cytotoxicity, although their anti-inflammatory activity was not investigated (5) .
Lipopolysaccharide (LPS)-activated macrophage cell lines are routinely used to evaluate the anti-inflammatory activities of natural and artificial phenolic antioxidants. Activation of macrophages by bacterial LPS induces local inflammation, antibody production, and other phenomena through the secretion of numerous proinflammatory cytokines, chemokines, and inflammatory mediators, such as reactive oxygen species (ROS) including nitric oxide (NO), as well as tumoricidal activity (6). Inhibition of cyclooxygenase-2 (COX2) expression is the major parameter used for assessing the effects of anti-inflammatory drugs, as COX2 is usually specific to inflamed tissues. Nuclear factor-kappa B (Nf-κB), a major transcription factor, is closely related to the expression of proinflammatory genes. The most abundant activated form of Nf-κB is a heterodimer composed of a p50 subunit and p65 subunit, which functions predominantly as a transcriptional activator (7, 8). Also, LPS is strong inducer of CD-40 expression in macrophages, which involves the activation of the transcription factor of Nf-κB (9).
In the present study, we investigated the inhibitory effects of p-cresol, p-cresol dimer, pHA, pHA dimer and BHA (Figure 1) on Cox2 expression in RAW264.7 cells stimulated by exposure to LPS. Since Nf-κb activation requires nuclear translocation of dimerized Nf-κb components such as p50, p52, p65 and RelB, we examined the inhibitory effect of these phenolic compounds on the nuclear translocation of each component in the presence of LPS.
Quantum chemical calculations can provide a more detailed insight into the molecular mechanisms underlying the biological activities of phenolic antioxidants. We previously demonstrated that honokiol and magnolol inhibit Porphyromonas gingivalis fimbria-stimulated Cox2 expression and also inhibit fimbria-stimulated Nf-κb activation in RAW264.7 cells. We also found that these compounds possessed high electronegativity (χ), suggesting a causal link between the anti-inflammatory activity of phenolic antioxidants and their χ value (10). These findings also suggest that quantum chemical calculations are useful for interpreting the mechanism responsible for the anti-inflammatory activity of phenolic antioxidants. Therefore, in the present study, we used the density functional theory (DFT) and B3LYP method with the 6-31G* basis set as a computational tool. The relationships between the anti-inflammatory activity of these compounds and their molecular properties, chemical hardness (η) and χ value, are discussed.
Materials and Methods
Materials. p-Cresol and pHA were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan. p-Cresol dimer and pHA dimer were synthesized from their monomers by the CuCl(OH)-catalyzed ortho coupling reaction described previously (11, 12). BHA was purchased from Tokyo Kasei Co. (Tokyo, Japan). The chemical structures of these phenol-related compounds are shown in Figure 1. Their solutions were prepared by dissolving each of them in dimethyl sulfoxide, and they were then diluted to the required concentrations using serum-free RPMI-1640 (Invitrogen Co., Carlsbad, CA, USA) as test samples. A Megaprime DNA labeling system, 5’-[α-32P]dCTP, and [γ-32P]ATP were purchased from Amersham Biosciences Co. (Piscataway, NJ, USA). A 5’-end labeling system was purchased from Promega Co. (Madison, WI, USA). A mouse Cox2 cDNA probe with a length of approximately 1.2 kbp was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). A 25-mer β-actin oligonucleotide (single-stranded DNA) probe was purchased from GeneDetect.com Ltd. (Bradenton, FL, USA). RPMI-1640 was purchased from Invitrogen Corp. Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). Escherichia coli O111 B4-derived LPS was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA).
Cell culture. The murine macrophage-like cell line RAW264.7, obtained from Dainippon Sumitomo Pharma Biomedical Co. Ltd. (Osaka, Japan), was used. The cells were cultured to a subconfluent state in RPMI-1640 medium supplemented with 10% FBS at 37°C and 5% CO2 in air, washed, and then incubated overnight in serum-free RPMI-1640. They were then washed again and treated with the test samples.
Cytotoxicity. The relative number of viable cells was determined using a Cell Counting Kit-8 (CCK-8) (Dojindo Co., Kumamoto, Japan) (13). In brief, RAW264.7 cells (3×104 per well) were cultured in NUNC 96-well plates (flat-well-type microculture plates) for 48 h, after which the cells were incubated with test samples for 24 h. CCK-8 solution was added to each well and then the absorbance was measured at 450 nm with a microplate reader (Biochromatic, Helsinki, Finland). The 50% lethal cytotoxic concentration (LC50) was determined from the dose–response curves. Data are expressed as the means of three independent experiments. Statistical analyses were performed using the Student's t-test.
Northern blot analysis and Cox2 mRNA inhibition. The procedure employed was similar to that reported previously (14). Briefly, 106 cells were placed in Falcon 5-cm-diameter dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and pre-treated for 30 min with each of the phenol-related compounds at a concentration of 10−6 M, 10−5 M or 10−4 M. They were then incubated in the presence or absence of LPS (100 ng/ml), and their total RNA was prepared 3 h later by the acid guanidine-phenol-chloroform procedure (15). The RNA was electrophoresed in 1% agarose gels with 0.2 M sodium phosphate as a running buffer, and then blotted onto nylon membranes (Micron Separations, Inc., Westboro, MA, USA). The membranes were then hybridized with a Cox2 cDNA probe labeled with 5’-[alpha-32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences Co.) and a β-actin oligonucleotide probe labeled with [γ-32P]ATP using a 5’-end labeling system purchased from Promega Co. After hybridization, the membranes were washed and dried, then exposed overnight to Kodak X-ray film (Carestram Health Inc., Rochester, NY, USA) at − 70°C. β-Actin was used as an internal standard for quantification of total RNA in each lane of the gel. Quantification of Cox2 expression was carried out by densitometry. The data were expressed as the relative signal intensity (percentage of maximum). Data are expressed as means of three independent experiments. Statistical analyses were performed using Student's t-test.
Inhibition of LPS-stimulated Cox2 mRNA expression by p-cresol, p-cresol dimer, pHA, pHA dimer and BHA in RAW264.7 cells was carried out as follows: The cells were pretreated for 30 min with or without addition of 10−5 M p-cresol, pHA, p-cresol dimer, pHA dimer or BHA, and then incubated with or without LPS at 100 ng/ml for 3 h. Total RNA was prepared and Cox2 mRNA expression was confirmed by northern blot analysis.
Preparation of nuclear extract and microwell colorimetric Nfκb assay. Nuclei were extracted and prepared for the microwell colorimetric Nfκb assay. In brief, the cells in 5-cm-diameter Falcon dishes (106 cells per dish) were pretreated for 30 min with or without the indicated doses of the compounds, and then treated with LPS at 100 ng/ml for 1 h. Thereafter, nuclear extracts were prepared using a Nuclear Extraction kit (Active Motif Co., Carlsbad, CA, USA) in accordance with the manufacturer's protocol. The microwell colorimetric Nfκb assay was performed as described previously (16) using the Trans-AM Nfκb family transcription factor assay kit (Active Motif Co.). Briefly, cell extracts were incubated in a 96-well plate coated with an oligonucleotide containing the Nfκb consensus binding site (5’-GGGACTTTCC-3’). Activated transcription factors from extracts that bound specifically to each respective immobilized oligonucleotide were detected in an enzyme-linked-immuno-sorbent assay (ELISA)-like assay using antibodies against the Nf-κb p50, p52, p65 and RelB subunits, followed by a secondary antibody conjugated to horseradish-peroxidase. Optical density was measured at 450 nm with a microplate reader (Biochromatic). The specificity of the assay was validated by including both the wild-type and mutated oligonucleotides in the reaction. Raji cell nuclear extract was used a positive control. Data are expressed as the means of three independent experiments. Statistical analyses were performed using Student's t-test.
Inhibition of LPS-stimulated Nf-κb activation by p-cresol, p-cresol dimer, pHA, pHA dimer or BHA in RAW264.7 cells was carried out as follows: The cells were pretreated for 30 min with or without addition of 10−5 M p-cresol, pHA, p-cresol dimer, pHA dimer or BHA, and then incubated with or without the LPS at 100 ng/ml for 1 h. Nuclear extracts were prepared and used in a TransAM (Active Motif) ELISA-like assay to quantify the Nfκb p50, p52, p65 and RelB DNA-binding activity.
Computation. The lowest unoccupied molecular orbital (LUMO) energy (ELUMO) and highest unoccupied molecular orbital (HOMO) energy (EHOMO) were obtained from ground state equilibrium geometries with density functional theory calculated DFT B3LYP 6-31G* in vacuum from 6-31G* initial geometry (10, 17).
The absolute value of HOMO energy was adopted as an approximate ionization potential (IP) value according to Koopman's theorem (18). All calculations were performed with Spartan'10 (Wave Function Inc., Irvine, CA, USA).
The η and χ values were calculated as follows: (Eq. 1) (Eq. 2)
Results
Cytotoxicity. The cytotoxicity of p-cresol, p-cresol dimer, pHA, pHA dimer and BHA towards RAW264.7 cells was investigated using CCK-8. The results are shown in Figure 2. The 50% lethal cytotoxic concentration (LC50) for p-cresol, p-cresol dimer, and pHA, pHA dimer and BHA was 850, 400, 1,100, 310 and 420 μM, respectively. pHA dimer showed the highest cytotoxicity, followed in order by p-cresol dimer and BHA. pHA exhibited the lowest cytotoxicity. The cytotoxicity of p-cresol dimer was double that of p-cresol monomer, whereas that for pHA dimer was three-times greater than for the pHA monomer.
Cox2 inhibition. Five compounds belonging to two representative chemical classes, p-cresol and the p-hydroxyanisole group, were tested at different concentrations up to 10−4 M with regard to their effect on Cox2 transcriptional activity in the presence of LPS. No significant decrease in cell viability was observed in cultures with LPS and the indicated phenols at a concentration of less than 10−4 M. The inhibitory effects of these compounds on LPS-stimulated Cox2 production in RAW264.7 cells were investigated within the concentration range of 10−6 M to 10−4 M. The results are shown in Figure 3. p-Cresol dimer and pHA dimer, particularly the former, dramatically inhibited the LPS-induced gene expression of Cox2 at 10−5 M. In contrast, the effective inhibitory activity for p-cresol and pHA was not attained at 10−5 M, but the inhibitory activity for p-cresol appeared to be greater than that for pHA. BHA exerted no inhibitory effect over a wide range of concentrations. Interestingly, no inhibitory activity was observed for any of the compounds tested at the high concentration of 10−4 M. This may have been attributable to the formation of intermediate phenolic compounds (quinones) through exposure to LPS; the intrinsic antioxidative properties of phenolic compounds may have decreased in the presence of mixtures of relatively high concentrations of phenols, radical scavengers and LPS that produce intercellular ROS. p-Cresol dimer and pHA dimer showed potent anti-inflammatory activity, whereas the activity of their monomers was weak. BHA had no activity.
Inhibition of Nf-κb activation and calculations. The results of inhibition of Nf-κb activation are shown in Figure 4, and the calculated HOMO, LUMO, η, χ and IP values for phenols are shown in TabIe I. As a reference compound, the calculated values of bis-BHA, a BHA dimer have been also added to TabIe I. This compound was previously reported to have potent anti-inflammatory activity (4). LPS-stimulated binding of Nfκb subunits such as p50, p52, p65 and RelB to its consensus sequences was markedly inhibited by p-cresol dimer and also by pHA dimer, but was weakly inhibited by monophenols, p-cresol and pHA, but not by BHA, as observed using an ELISA-like microwell colorimetric transcription factor assay kit. For each of p50, p52, p65 and RelB, the LPS-stimulated binding activity for p-cresol dimer was significantly greater than that for p-cresol at 10−6 M or 10−5 M. Similarly, the activity for pHA dimer was significantly lower than that for pHA at 10−6 M or 10−5 M. The LPS-stimulated binding of Nfκb was also completely inhibited by an excess amount of wild-type consensus oligonucleotide provided in the kit (data not shown). These findings indicated that p-cresol dimer and pHA dimer are potent inhibitors of LPS-triggered cellular signaling in RAW264.7 cells.
Quantum molecular properties. The results are shown in Table I. Molecules with a relatively large η value are generally unreactive, while those with a relatively small η value are generally reactive (17). pHA dimer and bis-BHA had small η values, possibly reflecting their bioactive nature. We previously reported a close relationship between phenol-induced cytotoxicity and η value for 2-methoxy phenols and 2-alkyl phenols, and found that their cytotoxicity increased as η decreased (19). pHA dimer and p-cresol dimer, with relatively small η values, had greater cytotoxicity than pHA or p-cresol. In contrast, the χ value declined in the order p-cresol dimer > pHA dimer > bis-BHA > p-cresol > pHA > BHA. The three dimers, p-cresol dimer, pHA dimer and bis-BHA, which possessed a high χ value, showed potent anti-inflammatory activity. It has been reported that bis-BHA has potent anti-inflammatory activity (20). The IP value declined in the order p-cresol dimer > p-cresol > pHA dimer > bis-BHA> pHA > BHA. Thus it is clear that BHA can act as a pro-oxidant, but not as an antioxidant, due to its small IP value.
Discussion
COX2 is the key enzyme that catalyzes the two sequential steps responsible for the biosynthesis of prostaglandins from arachidonic acid. COX2, the inducible isoform of COX, plays a critical role in renal function, maintenance of gastrointestinal integrity, ovarian and uterine function, bone metabolism, various inflammatory responses, rheumatoid arthritis, and Alzheimer's disease, and its overexpression has been associated with several types of pathologies, including neurodegenerative diseases and various types of cancers (10). The transcription of COX2 may be modulated by NF-κB, and the COX2 gene also contains numerous cis-acting promoter elements, including NF-κB sites (7), and NF-κB plays a critical role in mediating COX2 expression (21). NF-κB regulates the expression of genes involved in the control of proliferation/growth, inflammatory responses, cell adhesion, and other processes including other effector elements in a variety of cells (22). Functionally active NF-κB exists mainly as a heterodimer consisting of subunits of the Rel family (e.g. p50 p52, p65, RelB) which normally form an inactive cytoplasmic complex by binding to an inhibitory protein, IκB. Exposure of cells to external stimuli such as bacterial LPS, inflammatory cytokines and ROS is thought to cause rapid phosphorylation of IκB, with subsequent degradation by proteosomes. We previously found that honokiol and magnolol, phenolic antioxidants with potent anti-inflammatory activity, have a relatively large χ value, whereas eugenol, a 2-methoxy-4-allyl phenol that possesses no anti-inflammatory activity, had a smaller χ value than the former two compounds, suggesting that the anti-inflammatory activity was proportional to the difference in χ (10). Together with the findings of the present study, it can be assumed that the χ value of phenolic compounds controls their targeting enzymes and proteins involved in COX2 expression and NF-κB activation.
Putz et al. reported that the χ principle can be applied at the level of ligand-receptor binding in order to predict the genotoxicity and carcinogenicity of various chemicals (23). The molecular χ is first equalized with that of the receptor, leading to selection of a molecular fragment with χ complementary to that of the receptor, or adjustment of the receptor pocket to fit with the ligand χ. From these hypotheses it is assumed that COX2 enzyme and NF-κB proteins activated by inflammatory stimuli such as LPS and ROS may be controlled by the χ value of phenolic compounds. On the other hand, the radical-scavenging activity of phenolic compounds is well known to be related to their anti-inflammatory activity, as they can scavenge harmful free radicals. We previously reported that 2-tert-butylphenols, such as BHA, scavenged intracellular ROS (24). p-cresol dimer and pHA dimer also scavenged alkyl radicals to a much greater degree than p-cresol or pHA, as a result of the relatively high stoichiometric factor (number of free radicals trapped by one mole of phenolic antioxidant moiety) of these compounds: p-cresol dimer and pHA dimer scavenge about four radicals, whereas p-cresol and pHA scavenge about two radicals (5).
Furthermore, the IP is the most important energetic factor for evaluation of scavenging ability. It is known that a relatively high IP value decreases the rate of electron transfer between a phenolic antioxidant and oxygen, and therefore phenolic compounds with a higher IP value have reduced pro-oxidative potency, whereas compounds with a smaller IP value have stronger pro-oxidative properties (25). From these findings, it is assumed that BHA and pHA, with smaller IP values, may act as pro-oxidants. We have previously reported that BHA-induced cytotoxicity was enhanced by horseradish peroxidases due to pro-oxidant activity, suggesting that BHA is oxidizable and produces cytotoxic BHA radicals within the cell; consequently, cytotoxic quinone intermediates may be produced, and therefore BHA shows no anti-inflammatory activity (24). Quantum chemical calculations might provide a closer insight into the molecular mechanisms of anti-inflammatory activity of these phenolic compounds, and the η, χ and IP values may be useful for prediction of their modes of action.
- Received May 13, 2014.
- Revision received July 7, 2014.
- Accepted July 8, 2014.
- Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved