Synergistic induction of apoptosis in lung cancer cells through co-delivery of PLGA phytol/α-bisabolol nanoparticles.

This study explored the potential of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles to enhance the effectiveness of anticancer treatments through combination therapy with phytol and α-bisabolol. The encapsulation efficiency of the nanoparticles was investigated, highlighting the role of ionic interactions between the drugs and the polymer. Characterization of PLGA-Phy+Bis nanoparticles was carried out using DLS with zeta potential and HR-TEM for size determination. Spectrophotometric measurements evaluated the encapsulation efficiency, loading efficiency, and in vitro drug release. FTIR analysis assessed the chemical interactions between PLGA and the drug actives, ensuring nanoparticle stability. GC-MS was employed to analyze the chemical composition of drug-loaded PLGA nanocarriers. Cytotoxicity was evaluated via the MTT assay, while Annexin V-FITC/PI staining and western blot analysis confirmed apoptotic cell death. Additionally, toxicity tests were performed on L-132 cells and in vivo zebrafish embryos. The study demonstrates high encapsulation efficiency of PLGA-Phy+Bis nanoparticles, which exhibit monodispersity and sizes of 189.3±5nm (DLS) and 268±54 nm (HR-TEM). Spectrophotometric analysis confirmed efficient drug encapsulation and release control. FTIR analysis revealed nanoparticle structural stability without chemical interactions. MTT assay results demonstrated the promising anticancer potential of all the three nanoparticle types (PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis) against lung cancer cells. Apoptosis was confirmed through Annexin V-FITC/PI staining and western blot analysis, which also revealed changes in Bax and Bcl-2 protein expression. Furthermore, the nanoparticles exhibited non-toxicity in L-132 cells and zebrafish embryo toxicity tests. PLGA-Phy+Bis nanoparticles exhibited efficient encapsulation, controlled release, and low toxicity. Apoptosis induction in A549 cells and non-toxicity in healthy cells highlight their clinical potential.

Mice with a deficiency in Peroxisomal Membrane Protein 4 (PXMP4) display mild changes in hepatic lipid metabolism.

Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout (Pxmp4-/-) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4-/- mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4-/- mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4-/- mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids.

Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of ß-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1-/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1-/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1-/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1-/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Effects of dietary phytol on tissue accumulation of phytanic acid and pristanic acid and on the tissue lipid profiles in mice.

Recent in vitro evidence suggests that the phytol-derived fatty acids, phytanic acid (PA) and pristanic acid (PrA), are components of animal products with the potential to cause both beneficial and harmful effects on human health. In this study, we investigated the in vivo tissue accumulation of PA and PrA and the changes in tissue lipid profiles, using mice fed a phytol-containing diet. After 4 weeks of treatment with a diet containing 1.0% phytol, plasma, adipose tissue, liver, and brain were collected and their lipid profiles were biochemically and gas-chromatographically determined. Dietary phytol caused PA and PrA accumulation in the adipose tissue and liver but not in the brain, and reduced plasma and liver triacylglycerol levels. Phytol intake also decreased the fatty acid concentrations in the adipose tissue, especially polyunsaturated fatty acids such as linoleic acid, but increased the concentrations of these fatty acids in the liver. However, dietary phytol had a low impact on the brain lipid profile. This study suggests that dietary phytol intake caused accumulation of PA and PrA and modified lipid profiles in the adipose tissue and liver, but that the brain is an insusceptible tissue to dietary phytol-induced changes.

Phytol and its metabolites phytanic and pristanic acids for risk of cancer: current evidence and future directions.

This review summarizes the current evidence on the potential role of phytol, a microbial metabolite of chlorophyl A, and its metabolites, phytanic and pristanic acids, in carcinogenesis. Primary food sources in Western diets are the nut skin for phytol and lipids in dairy, beef and fish for its metabolites. Phytol and its metabolites gained interest as dietary compounds for cancer prevention because, as natural ligands of peroxisome proliferator-activated receptor-α and -γ and retinoid X receptor, phytol and its metabolites have provided some evidence in cell culture studies and limited evidence in animal models of anti-carcinogenic, anti-inflammatory and anti-metabolic-syndrome properties at physiological concentrations. However, there may be a narrow range of efficacy, because phytol and its metabolites at supra-physiological concentrations can cause in vitro cytotoxicity in non-cancer cells and can cause morbidity and mortality in animal models. In human studies, evidence for a role of phytol and its metabolites in cancer prevention is currently limited and inconclusive. In short, phytol and its metabolites are potential dietary compounds for cancer prevention, assuming the challenges in preventing cytotoxicity in non-cancer cells and animal models and understanding phytol metabolism can be mitigated.

A Phytol-Enriched Diet Activates PPAR-α in the Liver and Brown Adipose Tissue to Ameliorate Obesity-Induced Metabolic Abnormalities.

SCOPE: Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated transcription factor that regulates lipid and carbohydrate metabolism. We investigate the effects of naturally occurring PPAR-α agonists, phytol, and its metabolite phytanic acid, on obesity-induced metabolic disorders using a mouse model. METHODS AND RESULTS: A luciferase reporter assay shows that phytanic acid potently activates PPAR-α among PPAR subtypes. In high-fat-diet-induced, severely obese mice, a phytol-enriched diet increases phytanic acid levels in the liver and adipose tissue, where PPAR-α is abundantly expressed. A phytol-enriched diet ameliorates severe obesity and the related metabolic abnormalities of white adipose tissue. Moreover, the expression of PPAR-α target genes in the liver and brown adipose tissue is enhanced by a phytol-enriched diet, suggesting that phytol and phytanic acid activate PPAR-α in these organs. We confirm that phytanic acid treatment induced PPAR-α target gene expression in both primary hepatocytes and brown adipocytes from wild-type mice, but not in these cells from PPAR-α-deficient mice. CONCLUSION: A phytol-enriched diet may increase phytanic acid levels in the liver and brown adipocytes, thereby activating PPAR-α in these organs and ameliorating obesity-induced metabolic diseases.

Sex-dependent impact of Scp-2/Scp-x gene ablation on hepatic phytol metabolism.

While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid ¼ pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.

Effect of Fabp1/Scp-2/Scp-x Ablation on Whole Body and Hepatic Phenotype of Phytol-Fed Male Mice.

Liver fatty acid binding protein (Fabp1) and sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) genes encode proteins that enhance hepatic uptake, cytosolic transport, and peroxisomal oxidation of toxic branched-chain fatty acids derived from dietary phytol. Since male wild-type (WT) mice express markedly higher levels of these proteins than females, the impact of ablating both genes (TKO) was examined in phytol-fed males. In WT males, high phytol diet alone had little impact on whole body weight and did not alter the proportion of lean tissue mass (LTM) versus fat tissue mass (FTM). TKO conferred on dietary phytol the ability to induce weight loss as well as reduce liver weight, FTM, and even more so LTM. Concomitantly TKO induced hepatic lipid accumulation, preferentially threefold increased phospholipid (PL) at the expense of decreased triacylglycerol (TG) and total cholesterol. Increased PL was associated with upregulation of membrane fatty acid transport/translocase proteins (FATP 2,4), cytosolic fatty acid/fatty acyl-CoA binding proteins (FABP2, ACBP), and the rate limiting enzyme in PL synthesis (Gpam). Decreased TG and cholesterol levels were not attributable to altered levels in respective synthetic enzymes or nuclear receptors. These data suggest that the higher level of Fabp1 and Scp2/Scpx gene products in WT males was protective against deleterious effects of dietary phytol, but TKO significantly exacerbated phytol effects in males.

Synthesis and biological evaluation of C-3 aliphatic coumarins as vitamin K antagonists.

Since the discovery of Warfarin in the 1940s, the design of new warfarin-derived anticoagulants for rodent management has been challenging, with mainly structural modifications performed on the C3 position of the coumarin skeleton. In order to better understand the pharmacomodulation of such derivatives, we have synthesized a family of C3 (linear and branched) alkyl-4-hydroxycoumarins, which led to the identification of compounds 5e and 5f as potential short-term active anticoagulants.

Impact of dietary phytol on lipid metabolism in SCP2/SCPX/L-FABP null mice.

In vitro studies suggest that liver fatty acid binding protein (L-FABP) and sterol carrier protein-2/sterol carrier protein-x (SCP2/SCPx) gene products facilitate uptake and metabolism and detoxification of dietary-derived phytol in mammals. However, concomitant upregulation of L-FABP in SCP2/SCPx null mice complicates interpretation of their physiological phenotype. Therefore, the impact of ablating both the L-FABP gene and SCP2/SCPx gene (L-FABP/SCP2/SCPx null or TKO) was examined in phytol-fed female wild-type (WT) and TKO mice. TKO increased hepatic total lipid accumulation, primarily phospholipid, by mechanisms involving increased hepatic levels of proteins in the phospholipid synthetic pathway. Concomitantly, TKO reduced expression of proteins in targeting fatty acids towards the triacylglycerol synthetic pathway. Increased hepatic lipid accumulation was not associated with any concomitant upregulation of membrane fatty acid transport/translocase proteins involved in fatty acid uptake (FATP2, FATP4, FATP5 or GOT) or cytosolic proteins involved in fatty acid intracellular targeting (ACBP). In addition, TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass. Since individually ablating SCPx or SCP2/SCPx elicited concomitant upregulation of L-FABP, these findings with TKO mice help to resolve the contributions of SCP2/SCPx gene ablation on dietary phytol-induced whole body and hepatic lipid phenotype independent of concomitant upregulation of L-FABP.

Phytol has antibacterial property by inducing oxidative stress response in Pseudomonas aeruginosa.

Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.

Phytol, a diterpene alcohol, inhibits the inflammatory response by reducing cytokine production and oxidative stress.

Studies have shown that diterpenes have anti-inflammatory and redox-protective pharmacological activities. The present study aimed to investigate the anti-inflammatory properties of phytol, a diterpene alcohol, in a mouse model of acute inflammation, and phytol effect on leukocyte recruitment, cytokines levels, and oxidative stress. The anti-inflammatory activities of phytol were assessed by measuring paw edema induced by different inflammatory agents (e.g., λ-carrageenan, compound 48/80, histamine, serotonin, bradykinin, and prostaglandin E2 [PGE2 ]), myeloperoxidase (MPO) activity, peritonitis model and cytokine levels. Further, oxidative stress was evaluated by determining glutathione (GSH) levels and malondialdehyde (MDA) concentration. The results showed that phytol (7.5, 25, 50, and 75 mg/kg) significantly reduced carrageenan-induced paw edema, in a dose-dependent manner. In addition, phytol (75 mg/kg) inhibited compound 48/80-, histamine-, serotonin-, bradykinin- and PGE2 -induced paw edema. It also inhibited the recruitment of total leukocytes and neutrophils; decreased MPO activity, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels, and MDA concentration; and increased GSH levels during carrageenan-induced acute inflammation. These results suggest that phytol attenuates the inflammatory response by inhibiting neutrophil migration that is partly caused by reduction in IL-1ß and TNF-α levels and oxidative stress.

Anxiolytic-like effects of phytol: possible involvement of GABAergic transmission.

Phytol, a branched chain unsaturated alcohol, is particularly interesting because it is an isolated compound from essential oils of different medicinal plants. The aim of this study was to evaluate the anxiolytic-like effects of phytol in animal models to clarify their possible action mechanism. After acute intraperitoneal treatment with phytol at doses of 25, 50 and 75 mg/kg behavioral models of open-field, elevated-plus-maze, rota-rod, light-dark, marble-burying and pentobarbital sleeping time tests were utilized. In open field test, phytol (25, 50 and 75 mg/kg) [p<0.01] increased the number of crossings and rearings. However, the number of groomings [p<0.01] was reduced. Likewise, the number of entries and the time spent in light space were increased [p<0.01] while the number of marble-burying was decreased [p<0.001], in elevated-plus-maze, light-dark and marble-burying tests, respectively. In motor activity test, phytol (75 mg/kg) impaired the rota-rod performance of mice [p<0.01]. In pentobarbital sleeping time test, phytol 75 mg/kg decreased for latency of sleeping and phytol (25, 50 and 75 mg/kg) increased the sleep time when compared to negative control [p<0.05]. All these effects were reversed by pre-treatment with flumazenil (2.5mg/kg, i.p.), similarly to those observed with diazepam (2mg/kg, i.p.; positive control) suggesting that the phytol presents mechanism of action by interaction with the GABAergic system. These findings suggest that acute administration of phytol exerts an anxiolytic-like effect on mice. Furthermore, suppose that phytol interacts with GABAA receptor, probably at the receptor subtypes that mediate benzodiazepines effects, to produce sedative and anxiolytic activities.

Efficacy of phytol-derived diterpenoid immunoadjuvants over alum in shaping the murine host's immune response to Staphylococcus aureus.

The ubiquitous gram-positive bacterium Staphylococcus aureus occupies a unique niche in humans for its ability to survive both as a commensal and a life-threatening pathogen. Its complex relationship with the host and its ability to engender a throng of virulence factors, have hindered the development of a successful vaccine against it. The use of immunoadjuvants to enhance host immunity and prevent the shift from commensalism to pathogenicity is a rational approach for containing infection. The objective of this study was to understand the mechanisms by which alum and two phytol-derived immunoadjuvants, phytanol (PHIS-01)(1) and phytanyl chloride (PCl)(2) shape the interaction between S. aureus and its murine host. We studied the effects of the phytol derivatives, relative to alum, on the induction of inflammatory cytokines and chemokines, recruitment of CD11b(+) cells, generation of specific anti-S. aureus antibodies and in vitro clearance of S. aureus. Our results showed that both PHIS-01 and PCl were stronger inducers of protective cytokines IL-17 and IL-1ß than alum, and far exceeded alum in enhancing anti-S. aureus antibody response. However, both alum and the phytol derivatives (particularly PCl) promoted efficient recruitment of CD11b(+) cells. Furthermore, PHIS-01, alum and to a lesser extent, PCl were able to up-regulate the expression of key inflammation-related genes that were highly down-regulated by S. aureus alone. In vitro killing assays showed that both PHIS-01 and PCl were far more potent than alum in promoting S. aureus clearance; this indicated their efficiency in shaping an effective anti-S. aureus immune microenvironment. In summary, our study provides evidence for the better effectiveness of phytol-derived immunoadjuvants over alum in enhancing anti-S. aureus immunity.

Immune enhancement by novel vaccine adjuvants in autoimmune-prone NZB/W F1 mice: relative efficacy and safety.

BACKGROUND: Vaccines have profoundly impacted global health although concerns persist about their potential role in autoimmune or other adverse reactions. To address these concerns, vaccine components like immunogens and adjuvants require critical evaluation not only in healthy subjects but also in those genetically averse to vaccine constituents. Evaluation in autoimmune-prone animal models of adjuvants is therefore important in vaccine development. The objective here was to assess the effectiveness of experimental adjuvants: two phytol-derived immunostimulants PHIS-01 (phytanol) and PHIS-03 (phytanyl mannose), and a new commercial adjuvant from porcine small intestinal submucosa (SIS-H), relative to a standard adjuvant alum. Phytol derivatives are hydrophobic, oil-in water diterpenoids, while alum is hydrophilic, and SIS is essentially a biodegradable and collagenous protein cocktail derived from extracellular matrices. RESULTS: We studied phthalate -specific and cross-reactive anti-DNA antibody responses, and parameters associated with the onset of autoimmune disorders. We determined antibody isotype and cytokine/chemokine milieu induced by the above experimental adjuvants relative to alum. Our results indicated that the phytol-derived adjuvant PHIS-01 exceeded alum in enhancing anti-phthalate antibody without much cross reactivity with ds-DNA. Relatively, SIS and PHIS-03 proved less robust, but they were also less inflammatory. Interestingly, these adjuvants facilitated isotype switching of anti-hapten, but not of anti-DNA response. The current study reaffirms our earlier reports on adjuvanticity of phytol compounds and SIS-H in non autoimmune-prone BALB/c and C57BL/6 mice. These adjuvants are as effective as alum also in autoimmune-prone NZB/WF1 mice, and they have little deleterious effects. CONCLUSION: Although all adjuvants tested impacted cytokine/chemokine milieu in favor of Th1/Th2 balance, the phytol compounds fared better in reducing the onset of autoimmune syndromes. However, SIS is least inflammatory among the adjuvants evaluated.

Protective effect of pristane on experimental autoimmune uveitis.

This study evaluates the effects of pristane and phytol, two mineral oils with pro-oxidative effects, on the course of experimental autoimmune uveitis. C57BL6 mice were immunized with IRBP1-20 peptide emulsified in CFA and treated five days prior to immunization with phytol or with pristane or with PBS as control. Administration of pristane reduces the incidence and severity of IRBP-induced uveitis as demonstrated by the decrease in vasculitis and inflammatory foci in fundus and by a reduction in histological damages and leukocyte infiltration compared to untreated or phytol-treated mice. The protective effect observed is associated with a decreased activation of peripheral CD4+ and CD8+ T lymphocytes and a decrease in the intensity of the Th1 and Th17 autoimmune response to IRBP in pristane-treated mice compared to control mice, as evidenced by the decreased production of IFNγ and IL17 by IRBP-specific lymphocytes from lymph nodes draining the site of immunization and by the increased production of anti-IRBP IgG1 over IgG2a. In addition, HUVEC and ARPE-19 cells incubated with the sera of mice treated with pristane presented a reduced production of H(2)O(2). The benefit of lowering the systemic oxidative stress by pristane in the course of EAU was confirmed by injecting the antioxidant NAC in IRBP-immunized mice. As pristane, NAC decreased clinical and histological inflammation of the retina and preserved the integrity of the hemato-retinal barrier. Finally, the protective effect of pristane on the development of EAU suggests that some mineral oils may represent a new therapeutic strategy in human uveitis.

Synthetic adjuvants for vaccine formulations: evaluation of new phytol derivatives in induction and persistence of specific immune response.

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.

Phytol-induced hepatotoxicity in mice.

Phytanic acid is a branched-chain, saturated fatty acid present in high concentrations in dairy products and ruminant fat. Some other dietary fats contain lower levels of phytol, which is readily converted to phytanic acid after absorption. Phytanic acid is a peroxisome proliferator binding the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) to induce expression of genes encoding enzymes of fatty acid oxidation in peroxisomes and mitochondria. Administration of dietary phytol (0.5% or 1%) to normal mice for twelve to eighteen days caused consistent PPARalpha-mediated responses, such as lower body weights, higher liver weights, peroxisome proliferation, increased catalase expression, and hepatocellular hypertrophy and hyperplasia. Female mice fed 0.5% phytol and male and female mice fed 1% phytol exhibited midzonal hepatocellular necrosis, periportal hepatocellular fatty vacuolation, and corresponding increases in liver levels of the phytol metabolites phytanic acid and pristanic acid. Hepatic expression of sterol carrier protein-x (SCP-x) was five- to twelve-fold lower in female mice than in male mice. These results suggest that phytol may cause selective midzonal hepatocellular necrosis in mice, an uncommon pattern of hepatotoxic injury, and that the greater susceptibility of female mice may reflect a lower capacity to oxidize phytanic acid because of their intrinsically lower hepatic expression of SCP-x.

Ataxia with loss of Purkinje cells in a mouse model for Refsum disease.

Refsum disease is caused by a deficiency of phytanoyl-CoA hydroxylase (PHYH), the first enzyme of the peroxisomal alpha-oxidation system, resulting in the accumulation of the branched-chain fatty acid phytanic acid. The main clinical symptoms are polyneuropathy, cerebellar ataxia, and retinitis pigmentosa. To study the pathogenesis of Refsum disease, we generated and characterized a Phyh knockout mouse. We studied the pathological effects of phytanic acid accumulation in Phyh(-/-) mice fed a diet supplemented with phytol, the precursor of phytanic acid. Phytanic acid accumulation caused a reduction in body weight, hepatic steatosis, and testicular atrophy with loss of spermatogonia. Phenotype assessment using the SHIRPA protocol and subsequent automated gait analysis using the CatWalk system revealed unsteady gait with strongly reduced paw print area for both fore- and hindpaws and reduced base of support for the hindpaws. Histochemical analyses in the CNS showed astrocytosis and up-regulation of calcium-binding proteins. In addition, a loss of Purkinje cells in the cerebellum was observed. No demyelination was present in the CNS. Motor nerve conduction velocity measurements revealed a peripheral neuropathy. Our results show that, in the mouse, high phytanic acid levels cause a peripheral neuropathy and ataxia with loss of Purkinje cells. These findings provide important insights in the pathophysiology of Refsum disease.

Polyunsaturated fats attenuate the dietary phytol-induced increase in hepatic fatty acid oxidation in mice.

The effects of dietary phytol and the type of dietary fat on hepatic fatty acid oxidation were examined in male ICR mice. Mice were fed diets containing 0 or 5 g/kg phytol and 100 g/kg palm, safflower, or fish oil for 21 d. Among the groups fed phytol-free diets, the activities and mRNA abundance of various enzymes involved in fatty acid oxidation were greater in mice fed fish oil than in those fed palm or safflower oil. Dietary phytol profoundly increased the activities and mRNA abundance of hepatic fatty acid oxidation enzymes in mice fed palm oil. However, safflower and fish oils, especially the latter, greatly attenuated the phytol-dependent increase in hepatic fatty acid oxidation. The hepatic concentration of phytanic acid, a metabolite of phytol that is the ligand and activator of retinoid X receptors and peroxisome proliferator-activated receptors, was higher in mice fed fish oil than safflower or palm oil, and in those administered safflower oil than palm oil. The hepatic mRNA abundance of sterol carrier protein-2, a lipid-binding protein involved in phytol metabolism, was inversely correlated with the hepatic concentration of phytanic acid. We demonstrated that polyunsaturated fats attenuate the enhancing effect of dietary phytol on hepatic fatty acid oxidation. Dietary fat-dependent changes in the hepatic phytanic acid concentration cannot account for this phenomenon.