Theoretical basis validation and oxidative stress markers for cancer prevention clinical trials of aspirin.

Aspirin, a nonsteroidal anti-inflammatory drug, has been proven effective in a clinical trial of carcinogenesis blockade. However, various modes of action have been reported for these effects. Thus, in this study, we aimed to present reasonable mode of actions as a proof of concept for human trials, especially trials for patients with familial adenomatous polyposis (FAP). Aspirin treatment at 1000 ppm inhibited intestinal tumorigenesis in FAP model Min mice. As a mode of action, aspirin regulated ß-catenin signaling, inflammation, and oxidative stress both in vivo and in vitro. Furthermore, we examined novel markers predictive of aspirin treatment based on liquid biopsy. Here, we demonstrated that aspirin reduced the levels of reactive carbonyl species in the serum of Min mice. These data are expected to be of use for proof of concept of aspirin human trials and implied for the prediction of aspirin efficacy.

The anti-TLR4 monoclonal antibody Sa15-21 enhances inflammatory cytokine production in LPS-stimulated macrophages.

Sa15-21, a monoclonal antibody against mouse Toll-like receptor (TLR) 4, can protect mice from lipopolysaccharide (LPS)/D-galactosamine-induced acute lethal hepatitis. Herein, we investigated the molecular mechanisms underlying Sa15-21-mediated regulation of TLR4 signaling in macrophages. Results showed that Sa15-21 enhanced the production of proinflammatory cytokines and attenuated the production of anti-inflammatory cytokines in LPS-stimulated macrophages. Western blotting analysis revealed that Sa15-21 pretreatment had no effect on NF-κB and MAPK signaling in LPS-stimulated macrophages; however, Sa15-21 treatment alone led to a weak and delayed activation of NF-κB and MAPK signaling without any effect on proinflammatory cytokine production. By contrast, Sa15-21 failed to induce the activation of interferon regulatory factor 3. Taken together, our results indicate that Sa15-21 sensitizes macrophages to facilitate the inflammatory response via TLR signaling.

Cell surface expression of human RP105 depends on N-glycosylation of MD-1.

For its cell surface expression, radioprotective 105 (RP105) - an orphan Toll-like receptor - must form a complex with a soluble glycoprotein called myeloid differentiation 1 (MD-1). The number of RP105-negative cells is significantly increased in patients with systemic lupus erythematosus (SLE); however, to elucidate the mechanism underlying this increase, how RP105 is expressed on the cell surface depending on MD-1 should be investigated. We demonstrated that RP105 exhibits two forms depending on MD-1 and its two N-glycosylation sites, N96 and N156. Cell surface expression of RP105 decreased in the presence of mutant MD-1 (N96Q/N156Q). Nonglycosylated MD-1 decreased the de novo cell surface expression of RP105 but not pre-expressed RP105. Thus, the N-glycans of MD-1 may represent targets for SLE therapy.

Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study.

BACKGROUND: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure. METHODS: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice. RESULTS: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans. CONCLUSIONS: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

The Combination of Cigarette Smoking and Alcohol Consumption Synergistically Increases Reactive Carbonyl Species in Human Male Plasma.

Cigarette smoking and alcohol consumption are major risk factors for lifestyle-related diseases. Although it has been reported that the combination of these habits worsens risks, the underlying mechanism remains elusive. Reactive carbonyl species (RCS) cause chemical modifications of biological molecules, leading to alterations in cellular signaling pathways, and total RCS levels have been used as a lipid peroxidation marker linked to lifestyle-related diseases. In this study, at least 41 types of RCS were identified in the lipophilic fraction of plasma samples from 40 subjects using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Higher levels of 10 alkanals, 5 trans-2-alkenals, 1 cis-4-alkenal, and 3 alkadienals were detected in the smoking/drinking group (N = 10) as compared to those with either habit (N = 10 each) or without both habits (N = 10) in the analysis of covariances adjusted for age and BMI. The levels of 3 alkanals, 1 trans-2-alkenal, 1 alkadienal, and 1 4-hydroxy-2-alkenal in the smoking/drinking group were significantly higher than those in the no-smoking/drinking and no-smoking/no-drinking groups. These results strongly indicate that the combination of cigarette smoking and alcohol drinking synergistically increases the level and variety of RCS in the circulating blood, and may further jeopardize cellular function.

Clostridium butyricum MIYAIRI 588 Modifies Bacterial Composition under Antibiotic-Induced Dysbiosis for the Activation of Interactions via Lipid Metabolism between the Gut Microbiome and the Host.

The gut microbiome is closely related to gut metabolic functions, and the gut microbiome and host metabolic functions affect each other. Clostridium butyricum MIYAIRI 588 (CBM 588) upregulates protectin D1 production in host colon tissue following G protein-coupled receptor (GPR) 120 activation to protect gut epithelial cells under antibiotic-induced dysbiosis. However, how CBM 588 enhances polyunsaturated fatty acid (PUFA) metabolites remains unclear. Therefore, we focused on the metabolic function alterations of the gut microbiome after CBM 588 and protectin D1 administration to reveal the interaction between the host and gut microbiome through lipid metabolism during antibiotic-induced dysbiosis. Consequently, CBM 588 modified gut microbiome and increased the butyric acid and oleic acid content. These lipid metabolic modifications induced GPR activation, which is a trigger of ERK 1/2 signaling and directed differentiation of downstream immune cells in the host colon tissue. Moreover, endogenous protectin D1 modified the gut microbiome, similar to CBM 588. This is the first study to report that CBM 588 influences the interrelationship between colon tissue and the gut microbiome through lipid metabolism. These findings provide insights into the mechanisms of prevention and recovery from inflammation and the improvement of host metabolism by CBM 588.

Dietary Supplementation With Eicosapentaenoic Acid Inhibits Plasma Cell Differentiation and Attenuates Lupus Autoimmunity.

Accumulating evidence suggests that cholesterol accumulation in leukocytes is causally associated with the development of autoimmune diseases. However, the mechanism by which fatty acid composition influences autoimmune responses remains unclear. To determine whether the fatty acid composition of diet modulates leukocyte function and the development of systemic lupus erythematosus, we examined the effect of eicosapentaenoic acid (EPA) on the pathology of lupus in drug-induced and spontaneous mouse models. We found that dietary EPA supplementation ameliorated representative lupus manifestations, including autoantibody production and immunocomplex deposition in the kidneys. A combination of lipidomic and membrane dynamics analyses revealed that EPA remodels the lipid composition and fluidity of B cell membranes, thereby preventing B cell differentiation into autoantibody-producing plasma cells. These results highlight a previously unrecognized mechanism by which fatty acid composition affects B cell differentiation into autoantibody-producing plasma cells during autoimmunity, and imply that EPA supplementation may be beneficial for therapy of lupus.

The Radical Scavenger NZ-419 Suppresses Intestinal Polyp Development in Apc-Mutant Mice.

Colorectal cancer is the fourth leading cause of cancer death worldwide, and it is important to establish effective methods for preventing colorectal cancer. One effective prevention strategy could be the use of antioxidants. However, the role of the direct antioxidative function of antioxidants against carcinogenesis has not been clarified. Thus, we aimed to determine whether the direct removal of reactive oxygen species by a hydroxyl radical scavenger, NZ-419, could inhibit colorectal carcinogenesis. NZ-419 is a creatinine metabolite that has been shown to be safe and to inhibit the progression of chronic kidney disease in rats, and it is now under clinical development. In the present study, we demonstrated that NZ-419 eliminated reactive oxygen species production in HCT116 cells after H2O2 stimulation and suppressed H2O2-induced Nrf2 promoter transcriptional activity. The administration of 500 ppm NZ-419 to Apc-mutant Min mice for 8 weeks resulted in a decrease in the number of polyps in the middle segment of the small intestine to 62.4% of the value in the untreated control (p < 0.05 vs. control group). As expected, NZ-419 treatment affected the levels of reactive carbonyl species, which are oxidative stress markers in the serum of Min mice. Suppression of the mRNA levels of the proliferation-associated factor c-Myc was observed in intestinal polyps of Min mice after NZ-419 treatment, with a weak suppression of epithelial cell proliferation assessed by proliferation cell nuclear antigen (PCNA) staining in the intestinal polyps. This study demonstrated that NZ-419 suppress the development of intestinal polyps in Min mice, suggesting the utility of radical scavenger/antioxidants as a cancer chemopreventive agent.

Phospholipase A2 from bee venom increases poly(I:C)-induced activation in human keratinocytes.

Bee venom (BV) induces skin inflammation, characterized by erythema, blisters, edemas, pain and itching. Although BV has been found to have an inhibitory effect on toll-like receptors (TLRs), we here show that BV enhances keratinocyte responses to polyinosinic-polycytidylic acid [poly(I:C)], a ligand for TLR3. Our results revealed that the enhanced TLR activity was primarily induced by secretory phospholipase A2 (sPLA2), a component of BV (BV-sPLA2). PLA2 mediates the hydrolysis of membrane phospholipids into lysophospholipids and free fatty acids. We demonstrated that BV-sPLA2 increased the intracellular uptake of poly(I:C), phosphorylation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), and poly(I:C)-mediated interleukin 8 production in human keratinocytes. We further showed that the enzymatic activity of BV-sPLA2 was essential for the increased uptake of poly(I:C). These findings suggest that BV-sPLA2 may induce a modification of the cell membrane structure, leading to enhanced poly(I:C) uptake in keratinocytes. BV-sPLA2 might be able to promote wound healing by enhancing TLR3 responses.

Clostridium butyricum Modulates the Microbiome to Protect Intestinal Barrier Function in Mice with Antibiotic-Induced Dysbiosis.

Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that has previously been used to prevent antibiotic-associated diarrhea. However, the underlying mechanism by which CBM 588 protects the gut epithelial barrier remains unclear. Here, we show that CBM 588 increased the abundance of Bifidobacterium, Lactobacillus, and Lactococcus species in the gut microbiome and also enhanced the intestinal barrier function of mice with antibiotic-induced dysbiosis. Additionally, CBM 588 significantly promoted the expansion of IL-17A-producing γδT cells and IL-17A-producing CD4 cells in the colonic lamina propria (cLP), which was closely associated with changes in the intestinal microbial composition. Additionally, CBM 588 plays an important role in controlling antibiotic-induced gut inflammation through upregulation of anti-inflammatory lipid metabolites such as palmitoleic acid, 15d-prostaglandin J2, and protectin D1. This study reveals a previously unrecognized mechanism of CBM 588 and provides new insights into gut epithelial barrier protection with probiotics under conditions of antibiotic-induced dysbiosis.

A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza.

Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigen via the cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105 in vitro. To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

Sesame Lignans Suppress Age-Related Cognitive Decline in Senescence-Accelerated Mice.

Sesame lignans, which are biologically active compounds present in sesame seeds and oil, are known to have neuroprotective effects in several models of brain dysfunction. However, the effects of sesame lignans on age-related brain dysfunction are not clear and were thus investigated in the present study using a senescence-accelerated mouse (SAMP10). Two-month-old male SAMP10 mice were administrated a basal diet with 0% or 0.05% sesame lignans for two months, or with 0%, 0.02%, or 0.05% sesame lignans for 10 months and subjected to step-through passive avoidance tasks and forced swim tests. Reactive carbonyl species (RCs) were evaluated as markers of oxidative stress using a recently developed comprehensive analytical method. Both learning time in passive avoidance tasks and immobile time in forced swim tests became longer with aging (p < 0.05). However, the administration of sesame lignans significantly ameliorated age-related effects in both tests (p < 0.05). Age-related increases in RCs such as 4-hydroxy-2-nonenal in the cerebral cortex and liver were reduced in mice fed sesame lignans. These results suggest that sesame lignans can prevent age-related brain dysfunction via anti-oxidative activity.

Receptor-destroying enzyme (RDE) from Vibrio cholerae modulates IgE activity and reduces the initiation of anaphylaxis.

IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)-treated IgE cannot access its binding site on bone marrow-derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)-treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies.

Anticarcinogenic Effects of Dried Citrus Peel in Colon Carcinogenesis Due to Inhibition of Oxidative Stress.

Colorectal cancer is one of the leading causes of death worldwide. Reactive oxygen species produce oxidative stress and contribute to colorectal carcinogenesis. Because dietary citrus has been shown to reduce oxidative stress, we investigated the effects of citrus peel extract at dilutions of 1/200-1/500 on the activity of oxidative-stress-related transcription factors, including AP-1, NF-κB, NRF2, p53, and STAT3, in human colon cancer cell line HCT116 cells using a luciferase reporter gene assay. NRF2 transcriptional activities were 1.8- to 2.0-fold higher than the untreated control value. In addition, NF-κB, p53, and STAT3 transcriptional activities were 12-26% lower than the untreated control value. Administration of dried citrus peel in the diet of F344 rats at a dose of 1,000 ppm prevented the formation of azoxymethane-induced precancerous aberrant crypt foci (ACF) in the colon. The total number of ACF in rats fed with dried citrus peel was reduced to 75% of the control value. Moreover, the levels of oxidative-stress-related markers, reactive carbonyl species, in the serum of F344 rats were significantly reduced following the administration of dried citrus peel. These data suggest that citrus peel possesses an ability to suppress cellular oxidative stress through induction of NRF2, thereby preventing azoxymethane-induced colon carcinogenesis.

Chemopreventive effects of a low-side-effect antibiotic drug, erythromycin, on mouse intestinal tumors.

It is important to establish effective methods for preventing colorectal cancer because the number of colorectal cancer deaths is increasing. Erythromycin one of the macrolide antibiotics, has been shown to exert pleiotropic effects, such as anti-inflammatory and anti-oxidative effects, on mammalian cells. In the present study, we aimed to evaluate the preventive effects of erythromycin on intestinal carcinogenesis. We first confirmed that erythromycin suppresses the transcriptional activity of nuclear factor-κB and activator protein-1 and the expression of its downstream targets, interleukin-6 and cyclooxygenase-2 in human colon cancer cells. Next, we fed 5-week-old male Apc mutant Min mice with diets containing 500 ppm erythromycin for 15 weeks. Erythromycin treatment significantly reduced the number of proximal intestinal polyps to 70.9% of the untreated control value. Moreover, erythromycin reduced the levels of interleukin-6 and cyclooxygenase-2 mRNA expression in intestinal polyps. Although the levels of hepatic NADPH oxidase mRNA were decreased, erythromycin treatment did not affect the levels of oxidative stress markers, reactive carbonyl species, in the liver of Min mice. Our results suggest that erythromycin suppresses intestinal polyp development in Min mice, in part by attenuating local inflammation, and indicate that erythromycin is useful as a chemopreventive agent.

Metal-catalyzed oxidation of 2-alkenals generates genotoxic 4-oxo-2-alkenals during lipid peroxidation.

Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent adducts, which are associated with aging and disease processes. Since lipid peroxidation is a complex process and occurs in multiple stages, there might be yet unknown reaction pathways. Here, we analyzed comprehensively 2'-deoxyguanosine (dG) adducts with oxidized arachidonic acid using liquid chromatography-tandem mass spectrometry and found the formation of 7-(2-oxo-hexyl)-etheno-dG as one of the major unidentified adducts. The formation of this adduct was reproduced in the reaction of dG with 2-octenal and predominantly with 4-oxo-2-octenal (OOE). We also found that other 2-alkenals (with five or more carbons) generate corresponding 4-oxo-2-alkenal-type adducts. Importantly, it was found that transition metals enhanced the oxidation of C4-position of 2-octenal, leading to the formation of OOE-dG adduct. These findings demonstrated a new pathway for the formation of 4-oxo-2-alkenals during lipid peroxidation and might provide a mechanism for metal-catalyzed genotoxicity.

Irsogladine maleate, a gastric mucosal protectant, suppresses intestinal polyp development in Apc-mutant mice.

This study aimed to identify gastric mucosal protectants that suppress intestinal tumorigenesis in a mouse model. We chose six gastric mucosal protectants (ecabet sodium hydrate, irsogladine maleate, rebamipide, sofalcone, teprenone and troxipide) and examined their effects on the activity of oxidative stress-related transcriptional factors, including AP-1, NF-jB, NRF2, p53 and STAT3, in Caco-2 cells using a luciferase reporter gene assay. Among the six protectants, irsogladine maleate clearly inhibited NF-jB and AP-1 transcriptional activity. Furthermore, the chemopreventive property of irsogladine maleate was examined in a Min mouse model of familial adenomatous polyposis. Treatment with irsogladine maleate at doses of 5 and 50 ppm significantly reduced the number of intestinal polyps to 69% and 66% of the untreated control value, respectively. In these polyps, mRNA levels of the downstream targets of NF-jB, such as IL-1ß and IL-6, were decreased by irsogladine maleate treatment. Moreover, the levels of oxidative stress-related markers, reactive carbonyl species, in the livers of Min mice were clearly decreased following the administration of irsogladine maleate. This study demonstrated that irsogladine maleate suppresses intestinal polyp formation in Min mice partly through the NF-jB signaling pathway, thus reducing oxidative stress.

Comprehensive analysis of the lipophilic reactive carbonyls present in biological specimens by LC/ESI-MS/MS.

A new analytical method has been developed for profiling lipophilic reactive carbonyls (RCs) such as aldehydes and ketones in biological samples using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with selected reaction monitoring (SRM). The method consists of several phases, including (1) extraction of lipophilic RCs with a chloroform/methanol mixture; (2) derivatization of the extracted RCs with dansyl hydrazine (DH); and (3) SRM detection of the characteristic product ion of the 5-dimethylaminonaphthalene-1-sulfonyl moiety (m/z 236.1). The analytical results were expressed as RC maps, which allowed for the occurrence and levels of different lipophilic RCs to be visualized. We also developed a highly reproducible and accurate method to extract, purify and derivatize RCs in small volumes of biological specimens. This method was applied to the detection of free RCs in mice plasma samples, and resulted in the detection of more than 400 RCs in samples obtained from C57BL/6J mice. Thirty-four of these RCs were identified by comparison with authentic RCs. This method could be used to investigate the levels of RCs in biological and environmental samples, as well as studying the role of lipid peroxidation in oxidative stress related-disorders and discovering new biomarkers for the early diagnosis of these diseases.

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases.

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C7 having a relatively weak carbon-hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen ((1)ΔgO2) and ozone (O3) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5ß-, 6α- and 6ß-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ5,6 -double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.

Cytotoxic effects of secosterols and their derivatives on several cultured cells.

The cytotoxic effects of various oxysterols on several culture cells were examined. Ozonolysis products of cholesterol, secosterols (3ß-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product (3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde) and their keto alcohol and acid derivatives, were found to have potent cytotoxic activities, as compared with major endogenous oxysterols such as 5ß,6ß-epoxycholesterol, 7ß-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol. Secosterols might play important roles in tissue damage and inflammation-associated diseases.