Sequential Intercellular Delivery Nanosystem for Enhancing ROS-Induced Antitumor Therapy.

Despite recent advances in enhancing photodynamic therapy efficacy, high-efficiency reactive oxygen species (ROS)-based therapy approach, especially in malignancy tumor treatment, remains challenging. Relieving the hypoxia of tumor tissue has been considered to be an attractive strategy for enhancing ROS-based treatment effect. Nevertheless, it is frequently neglected that the hypoxic regions are usually located deep in the tumors and therefore are usually inaccessible. To address these limitations, herein we constructed a sequential intercellular delivery system (MFLs/LAOOH@DOX) that consists of a membrane fusion liposomes (MFLs) doped with linoleic acid hydroperoxide (LAOOH) in the lipid bilayer and antitumor doxorubicin (DOX) encapsulated inside. In this report, LAOOH, one of the primary products of lipid peroxidation in vivo, was selected as ROS-generated agent herein, which depends on Fe2+ rather than oxygen and other external stimuli to produce ROS. Upon the enhanced permeation and retention effect, MFLs/LAOOH@DOX first fused with tumor cell membranes in the perivascular region in synchrony with selective delivery of LAOOH into the plasma membrane and the on-demand intracellular release of DOX. By hitchhiking with extracellular vesicles, LAOOH, as a cell membrane natural ingredient, spread gradually to neighboring cells and throughout the entire tumor eventually. Combined with subsequent administration of nano Fe3O4, ROS was specifically generated on the tumor cell membrane by LAOOH throughout the tumor tissues. This study offers a new method to enhance ROS-based antitumor treatment efficiency.

Investigation of the role of 8-OHdG and oxidative stress in papillary thyroid carcinoma.

The aim of this study was to determine levels of serum 8-hydroxy-2'-deoxyguanosine (8-OHdG) as an indicator of oxidant-induced DNA damage and oxidant status in patients with papillary thyroid carcinoma before and after surgery. This study included 25 patients with papillary thyroid carcinoma and age-matched 27 healthy controls. Total antioxidant status (TAS), total oxidant status (TOS), lipid hydroperoxide (LOOH), and 8-OHdG levels were measured. 8-OHdG levels were significantly higher in the preoperative papillary thyroid carcinoma (PTC) group compared with the healthy control group (p < 0.001) and were significantly lower after operation in patients with papillary thyroid carcinoma (p = 0.004). Oxidative stress index (OSI) levels were significantly higher in both preoperative and postoperative PTC patients compared with the healthy control group (p < 0.001 and p < 0.001, respectively). TOS levels were higher in the preoperative and postoperative PTC groups compared to the healthy control group (p < 0.001 and p < 0.001, respectively). TAS levels was lower in the preoperative PTC groups compared to the healthy control group (p = 0.011). Serum LOOH levels were higher in both preoperative and postoperative PTC groups compared to the healthy control group (p < 0.001 and p < 0.001, respectively). Correlation analysis yielded that serum 8-OHdG levels were positively correlated with OSI and LOOH levels in patients with PTC before surgery (r = 0.668, p < 0.001; r = 0.446, p = 0.025, respectively) and had a negative correlation with TAS levels (r = -0.616, p = 0.001). We have shown severe oxidative DNA damage and impaired antioxidant status in papillary thyroid carcinoma.

L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.

To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5-HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants.

Association of elevated blood pressure and impaired vasorelaxation in experimental Sprague-Dawley rats fed with heated vegetable oil.

BACKGROUND: Poor control of blood pressure leads to hypertension which is a major risk factor for development of cardiovascular disease. The present study aimed to explore possible mechanisms of elevation in blood pressure following consumption of heated vegetable oil. METHODS: Forty-two male Sprague-Dawley rats were equally divided into six groups: Group I (control)--normal rat chow, Group II--fresh soy oil, Group III--soy oil heated once, Group IV--soy oil heated twice, Group V--soy oil heated five times, Group VI--soy oil heated ten times. Blood pressure was measured at the baseline level and at a monthly interval for six months. Plasma nitric oxide, heme oxygenase and angiotensin-converting enzyme levels were measured prior to treatment, at month-three and month-six later. At the end of treatment, the rats were sacrificed and thoracic aortas were taken for measurement of vascular reactivity. RESULTS: Blood pressure increased significantly (p<0.01) in the repeatedly heated oil groups compared to the control and fresh soy oil groups. Consumption of diet containing repeatedly heated oil resulted higher plasma angiotensin-converting enzyme level and lower nitric oxide content and heme oxygenase concentration. Reheated soy oil groups exhibited attenuated relaxation in response to acetylcholine or sodium nitroprusside, and greater contraction to phenylephrine. CONCLUSION: As a result of consumption of repeatedly heated soy oil, an elevation in blood pressure was observed which may be due to the quantitative changes in endothelium dependent and independent factors including enzymes directly involved in the regulation of blood pressure.

Monocarboxylate transporter 1 and CD147 are up-regulated by natural and synthetic peroxisome proliferator-activated receptor alpha agonists in livers of rodents and pigs.

Monocarboxylate transporter (MCT)-1 mediates the transport of ketone bodies and other monocarboxylic acids across the plasma membrane. MCT1 is up-regulated by peroxisome proliferator-activated receptor (PPAR)-alpha, a transcription factor that mediates the adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketogenesis. Here, we show for the first time that MCT1 is up-regulated by dietary natural PPAR-alpha agonists. Both, an oxidized fat and conjugated linoleic acids increased MCT1 mRNA concentration in the liver of rats. Also, in the liver of pigs as non-proliferating species MCT1 was up-regulated upon PPAR-alpha activation by clofibrate, oxidized fat and fasting. Concomitant with up-regulation of MCT1, mRNA level of CD147 was increased in livers of rats and pigs. CD147 is a plasma membrane glycoprotein that is required for translocation and transport activity of MCT1. CD147 mRNA increase upon PPAR-alpha activation could not be observed in mice lacking PPAR-alpha, which also fail in up-regulation of MCT1 indicating a co-regulation of MCT1 and CD147. Analysis of the 5'-flanking region of mouse MCT1 gene by reporter gene assay revealed that promoter activity of mouse MCT1 was not induced by PPAR-alpha, indicating that the 5'-flanking region is not involved in MCT1 regulation by PPAR-alpha.

Astaxanthin protects neuronal cells against oxidative damage and is a potent candidate for brain food.

Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. Based on the report claiming that AST could cross the brain-blood barrier, the aim of this study was to investigate the neuroprotective effect of AST by using an oxidative stress-induced neuronal cell damage system. The treatment with DHA hydroperoxide (DHA-OOH) or 6-hydroxydopamine (6-OHDA), either of which is a reactive oxygen species (ROS)-inducing neurotoxin, led to a significant decrease in viable dopaminergic SH-SY5Y cells by the MTT assay, whereas a significant protection was shown when the cells were pretreated with AST. Moreover, 100 nM AST pretreatment significantly inhibited intracellular ROS generation that occurred in either DHA-OOH- or 6-OHDA-treated cells. The neuroprotective effect of AST is suggested to be dependent upon its antioxidant potential and mitochondria protection; therefore, it is strongly suggested that treatment with AST may be effective for oxidative stress-associated neurodegeneration and a potential candidate for natural brain food.

Dietary ALEs are a risk to human health--NOT!

Advanced lipoxidation end-products (ALEs) are formed by reaction of protein with lipid-derived reactive peroxyl and carbonyl compounds produced during food processing and cooking. There is concern that ALEs may induce damage in the gastrointestinal tract, affecting gut health, or enter the body and promote vascular inflammation and tissue damage. However, there is no direct evidence that ALE-proteins are a source of damage in the intestines or that they are transported into the circulation and cause pathology. Modification of proteins by ALEs impedes their digestion, and reactive ALEs released by gastrointestinal proteases would react with proteins or peptides in the gut, limiting their absorption. There are also potent enzymatic mechanisms for detoxifying ALEs or their precursors prior to their entry into the circulation. If ALEs gain access to the circulation, a battery of protective enzymes in tissue provides a second level of defense. These enzymes may be induced in intestinal epithelia and liver by low doses of ALEs, and adaptive responses would provide enhanced protection against future exposure to ALEs. Overall, except in persons with compromised organ function, e. g., vascular, hepatic, or renal diseases, there is little evidence that food ALEs will have any significant pathological effects.

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: implication of their membrane fluidizing action.

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called "green notes". The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.

Suppression of tumour-promoting factors in fat-induced colon carcinogenesis by the antioxidants caroverine and ubiquinone.

Fatty acid hydroperoxides are produced from unsaturated fatty acids in the presence of oxygen at elevated temperatures during food processing. Their effects on gene expression in colorectal tumour cells were studied using linoleic acid hydroperoxide (LOOH) as a model compound. Addition of LOOH to the medium of LT97 adenoma and SW480 carcinoma cells enhanced the production of hydrogen peroxide. Both cell lines were observed to increase VEGF factors based on mRNA. High consumption of dietary fat promotes colon carcinogenesis in the long-term. While this effect is well known, the underlying mechanisms are not understood. An approach was made starting from the assumption that LOOH is present in dietary fats as a result of heating. LOOH undergoes homolytic cleavage in the presence of iron. Various radicals are formed on mixing LT97 or SW480 cells with LOOH. The expression of tumour-promoting factors was inhibited by caroverine and ubiquinone, which may be justified as active chemopreventive agents.

Diet high in lipid hydroperoxide by vitamin E deficiency induces insulin resistance and impaired insulin secretion in normal rats.

To clarify the effect of dietary lipid hydroperoxide (LPO) on development of glucose intolerance, we fed Sprague-Dawley rats on a diet containing elevated LPO level for 10 weeks and measured both insulin sensitivity and insulin secretion. The contents of LPO in both plasma and skeletal muscle in the LPO-fed rats were significantly higher than those in the controls. Both insulin resistance evaluated by steady-state blood glucose (SSBG) methods and impaired insulin secretion evaluated by oral glucose tolerance test (OGTT) were found in the LPO-fed rats as compared with control rats. Furthermore, the levels of insulin receptor substrate (IRS)-1 protein in the skeletal muscle were significantly lower in the LPO-fed rats. Those impairments were not reversed in LPO-fed rats with supernormal levels of plasma vitamin E following vitamin E supplementation for 5 weeks. Moreover, the immunohistochemical study revealed that NF-kappaB-p50 protein was found in the nucleus of pancreatic beta-cells of the LPO-fed rats, whereas it was not observed in the nucleus of the islets in the control rats. These findings indicate that NF-kappaB is activated in response to oxidative stress in pancreatic islet cells in LPO-fed rats. In conclusion, our studies reveal that diet high in LPO by vitamin E deficiency accelerates glucose intolerance through impairments of both sensitivity and secretion of insulin.

Chronic lipid hydroperoxide stress suppresses mucosal proliferation in rat intestine: potentiation of ornithine decarboxylase activity by epidermal growth factor.

We recently demonstrated that chronic exposure of rat intestine to sublethal levels of peroxidized lipids suppresses ornithine decarboxylase (ODC) activity, consistent with attenuated intestinal proliferation. The current study was designed to better understand the influence of exogenous epidermal growth factor (EGF) on intestinal proliferation in normal intestine and intestine that was challenged by oxidative stress induced by dietary consumption of peroxidized lipids. Male Sprague-Dawley rats (250-300 g) were fed standard chow (control) or peroxidized lipid chow for 4 weeks. EGF was injected intraperitoneally at a dose of 40 microg/kg. Intestinal proliferation was evaluated by ODC activity in fed or fasted states and at specified times during the circadian phase. Chronic peroxide consumption significantly attenuated ODC activities in association with increased tissue peroxide content. The suppressed ODC activities were restored to control values by EGF in the small intestine; in the colon, EGF increased ODC activity threefold over control rats given EGF. This elevated colonic ODC activity was correlated with decreased tissue GSSG and an increased GSH/GSSG ratio. These results show that EGF administration reverses the suppression of intestinal ODC activities induced by chronic peroxidized lipid intake. In contrast, EGF significantly elevates proliferative activity in the peroxide-stressed colon. This exaggerated proliferation may contribute to a better understanding of colonic susceptibility to oxidant-induced malignant transformation.

Lipid hydroperoxide stimulates subretinal choroidal neovascularization in the rabbit.

The authors sought to evaluate the effect of linoleic acid hydroperoxide (18:2/LHP) in promoting choroidal neovascularization (CNV). Albino male rabbits received a subretinal injection of various amounts of LHP (ranging from 5 to 200 microg) dissolved in 50 microl of sodium borate buffer. Control eyes received the buffer only. Eyes were examined up to 4 weeks later with indirect ophthalmoscopy, fundus photography and fluorescein angiography. Animals were killed on days 3, 7, 14 or 28, and eyes examined by light and transmission electron microscopy. In eyes injected with LHP of 150-200 microg, exposed areas turned white as observed ophthalmoscopically and showed both severe retinal and choroidal atrophy histologically. Neither fluorescein leakage nor CNV was found in these eyes or in controls. In 33 eyes injected with LHP of 100 microg or less, prominent fluorescein leakage was seen in three (9%) and less prominent focal leakage in five (15%). In 11 (46%) of the 24 eyes injected with 12.5-50 microg LHP, CNV was found histologically. Subretinal injection of LHP is capable of inducing CNV.

Catabolic fate of dietary trilinoleoylglycerol hydroperoxides in rat gastrointestines.

To elucidate whether dietary lipid peroxides are absorbed in the body, the catabolic fate of trilinoleoylglycerol hydroperoxides (TL-OOH), in the gastrointestines of rats was examined. Oxidized trilinoleoylglycerol with a peroxide value of 1000 meq/kg, 0.5 or 20 mg, was dosed intragastrically to rat together with 59.5 or 40 mg unoxidized trilinoleoylglycerol, respectively. The fate of TL-OOH in gastric and intestinal lumina was determined by high-performance liquid chromatography periodically until 240 min after treatment. At low dose, TL-OOH was soon broken down to linoleic acid hydroperoxides (LA-OOH) and hydroxyls, probably through gastric lipases, whereas at high dose, TL-OOH was retained in the stomach. In both cases, TL-OOH did not reach the intestines, though the unoxidized lipids moved to the intestines. When LA-OOH was given intragastrically, the lipids decomposed in the stomach, and linoleic acid hydroxyls, hexanal, 9-oxononanoic acid, and two novel compounds were detected 30 min after treatment. The novel compounds were identified to be epoxyketones, 11-oxo-12,13-epoxy-9- and 11-oxo-9,10-epoxy-12-octadecenoic acids. Thus, dietary TL-OOH was broken down in the stomach releasing, LA-OOH which decomposed further, and did not reach the intestines.

Dietary hydroperoxides of linoleic acid decompose to aldehydes in stomach before being absorbed into the body.

Our previous study (Biochim. Biophys. Acta 1393 (1998) 336-348, this issue) found that dietary hydroperoxides of trilinoleoylglycerol were broken down, releasing linoleic acid hydroperoxides (LA-OOH) in the stomach without reaching the intestines. The present paper describes the catabolic fate of LA-OOH in rat gastrointestines, in an attempt to elucidate those products which can be absorbed into the body. At an intragastric dose of 6.5 or 18 mumol, LA-OOH was not transported to the intestines as determined by HPLC. At large doses (200 or 800 mumol), much greater than that in the daily diet, there was partial leakage of LA-OOH to the intestines. The periodical fate was analyzed with 17.2 mumol [U-14C]LA-OOH chemically and radiochemically. Exemplifying the product composition at 30 min after treatment (as percentage of dosed amount), 27% unchanged LA-OOH, 9.7% epoxyketones, 3.5% hydroxyls (LA-OH), 2.4% decomposed aldehydes, and 13% unknown products were found in the gastric lumen. Another 25% was incorporated in the gastric tissue, and the other 6.4% occurred in the intestinal lumen and tissue as decomposed aldehyde. The LA-OH further decomposed to aldehydes with time in the stomach. When an aldehyde mixture was prepared and dosed, significant increases in hexanal and 4-hydroxynonenal were detected in the liver 15 h later. These results show that the dietary LA-OOH is decomposed to aldehydes in the stomach and that aldehydes are partly absorbed into the body.

Oxidized lipids in the diet are incorporated by the liver into very low density lipoprotein in rats.

Previous studies have shown that the quantity of oxidized lipids in the diet directly correlates with the level of oxidized chylomicrons in mesenteric lymph and the level of oxidized lipids in endogenous lipoproteins such as very low density lipoprotein (VLDL) and low density lipoprotein (LDL). The aim of the present study was to determine whether oxidized fatty acids in the diet are delivered via chylomicrons to the liver and whether these lipids are repackaged and secreted in VLDL. In these experiments, oxidized [14C]linoleic acid was utilized as a marker for oxidized dietary fats. When we determined the metabolism of nonoxidized and oxidized [14C]linoleic acid-labeled chylomicrons, we found that hepatic uptake was similar with 13.57 +/- 0.84% of nonoxidized and 13.40 +/- 0.96% of oxidized linoleic acid delivered to the liver 30 min after chylomicron administration. Additionally, uptake by the extrahepatic tissues was also similar. When the hepatic secretion of VLDL was determined in an in vitro perfusion system after the administration of nonoxidized and oxidized linoleic acid-labeled chylomicrons to intact animals, we found that oxidized linoleic acid was utilized for the formation and secretion of VLDL. After the administration of labeled nonoxidized and oxidized linoleic acid, 0.86 +/- 0.07% and 0.70 +/- 0.09% of the administered label was found in the liver perfusate at 2 h, respectively. The presence of oxidized linoleic acid in oxidized VLDL was confirmed by demonstrating the presence of hydroperoxide-derived hydroxy octadecanoic acid. Thus, our findings demonstrate that oxidized dietary lipids are delivered to the liver via chylomicrons where they are utilized for synthesis of endogenous lipoproteins such as VLDL.

Cisplatin-induced ototoxicity: the effect of pigmentation and inhibitory agents.

Cis-diamminedichloroplatinum II (cisplatin), a divalent platinum compound and potent cell-cycle nonspecific chemotherapeutic agent, produces a dose-limiting, permanent, high-frequency sensori-neural hearing loss and peripheral neuropathy, and a dose-related cumulative renal insufficiency with tubular necrosis and interstitial nephritis. The potential for dose-limiting and permanent cochlear (neuro) toxicity remains despite present methods of hypertonic saline, prehydration, and mannitol diuresis prior to drug administration. The exact mechanism(s) of ototoxicity and/or nephrotoxicity are still unknown. Continued aggressive high-dose cisplatin chemotherapy necessitates the investigation of ways to decrease the dose-limiting side effects that inhibit the administration of cisplatin at therapeutic and tumoricidal doses. This multifaceted project investigates two categories of potential inhibitors of cisplatin toxicity that, when coadministered with a known tumoricidal and ototoxic dose of cisplatin, will decrease or inhibit the ototoxicity: 1. phosphonic acid antibiotics (fosfomycin; 1,2 epoxypropylphosphonic acid); 2. nonglucocorticoid 21-aminosteroids, which are free oxygen radical scavengers (LAZAROIDS: U74006F and U78517F). This project also investigates the role of pigmentation as a variable affecting the evaluation of platinum-induced ototoxicity in the guinea pig animal model. Identification of an optimal animal model for future cisplatin toxicity research should be based on previously established species-specific differences in total drug dose, systemic toxicity, and morphological and functional evidence of cochlear toxicity, as affected by differences in pigmentation and drug tolerance. Cytocochleography, brainstem auditory evoked response (BSER), scanning and transmission electron microscopy of organ of Corti and the stria vascularis, double-blind light microscopy of renal, small intestine, and peripheral nerve tissue, and gamma-emission analysis of 195Mplatinum localization in inner ear neuroepithelium and the stria vascularis are used in the global evaluation of the ototoxic effects of cisplatin in both the adult albino and pigmented guinea pig.

Distribution of 14C after oral administration of [U-14C]labeled methyl linoleate hydroperoxides and their secondary oxidation products in rats.

To study the toxicity of low molecular weight (LMW) compounds formed during the autoxidation of oils, 14C-labeled primary monomeric compounds (methyl linoleate hydroperoxides) and secondary oxidation products, i.e., polymer and LMW compounds prepared from autoxidized methyl [U-14C]linoleate hydroperoxides (MLHPO) were orally administered to rats, and their radioactive distributions in tissues and organs were compared. The polymeric fraction consisted mainly of dimers of MLHPO. For the LMW fraction, 4-hydroxy-2-nonenal, 8-hydroxy methyl octanoate and 10-formyl methyl-9-decenoate were identified as major constituents by gas chromatography-mass spectrometry (GC-MS) after chemical reduction and derivatization. When LMW compounds were administered to rats, 14CO2 expiration and the excreted radioactivity in urine in 12 hr were significantly higher than those from polymer or MLHPO administration. Maximum 14CO2 expiration appeared 2-4 hr after the dose of LMW compounds. Radioactivity of the upper part of small intestines six hr after the dose of LMW compounds was higher than the values from administered polymer or MLHPO. The remaining radioactivity in the digestive contents and feces 12 hr after administration of LMW compounds was much lower than the values observed from administered polymer or MLHPO. Among internal organs, the liver contained the highest concentration of radioactivities from polymer, MLHPO and LMW fractions, and an especially higher level of radioactivity was found in liver six hr after the administration of LMW compounds. Six hours after the dose of LMW compounds, a relatively higher level of radioactivity also was detected in kidney, brain, heart and lung.(ABSTRACT TRUNCATED AT 250 WORDS)