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There is an ongoing need to do more with less and provide highly multiplexed analysis from limited sample volumes. Improved "sample sparing" assays would have a broad impact across pediatric and other rare sample type studies in addition to enabling sequential sampling. This capability would advance both clinical and basic research applications. Here we report the micro blood analysis technology (μBAT), a microfluidic platform that supports multiplexed analysis of neutrophils from a single drop of blood. We demonstrate the multiplexed orthogonal capabilities of μBAT including functional assays (phagocytosis, neutrophil extracellular traps, optical metabolic imaging) and molecular assays (gene expression, cytokine secretion). Importantly we validate our microscale platform using a macroscale benchmark assay. μBAT is compatible with lancet puncture or microdraw devices, and its design facilitates rapid operations without the need for specialized equipment. μBAT offers a new method for investigating neutrophil function in populations with restricted sample amounts.

Background: Prostatic cancers include a diverse microenvironment of tumor cells, cancer-associated fibroblasts, and immune components. This tumor microenvironment (TME) is a known driving force of tumor survival after treatment, but the standard-of-care tissue freezing or fixation in pathology practice limit the use of available approaches/tools to study the TME's functionality in tumor resistance. Thus, there is a need for approaches that satisfy both clinical and laboratory endpoints for TME study. Here we present methods for clinical case identification, tissue processing, and analytical workflow that are compatible with standard histopathology while enabling molecular and functional interrogation of prostate TME components., Methods: We first performed a small retrospective review to identify cases where submission of alternate prostate tissue slices and a parallel live tissue processing protocol complement traditional histopathology and enable viable multicompartment analysis of the TME. Then, we tested its compatibility with commonly employed methods to study the microenvironment including quantification of components both in situ and after tissue dissociation. We also evaluated tissue digestion conditions and cell isolation techniques to aid various molecular and functional endpoints., Results: We identified Gleason Grade Group 3+ clinical cases where tumor volume was sufficient to allow slicing of unfixed tissue and distribution of alternating tissue slices to standard-of-care histopathology and viable multi-modal TME analyses. No single method was found that preserved cellular sub-types for all downstream readouts; instead, tissues were further divided so techniques could be catered to each endpoint. For instance, we show that incorporating the protease dispase into tissue dissociation improves viability for culture and functional analyses but hinders immune cell analysis by flow cytometry. We also found that flow activated cell sorting provides highly pure cell populations for quantitative reverse-transcription polymerase chain reaction and RNA-seq while isolation using antibody-labeled paramagnetic particles facilitated functional coculture experiments., Conclusions: The identification of candidate cases and use of these techniques enable translational research and the development of molecular and functional assays to facilitate prostate TME study without compromising standard-of-care histopathological diagnosis. This allows bridging clinical histopathology and further interrogation of the prostate TME and promises to advance our understanding of tumor biology and unveil new predictive and prognostic markers of prostate cancer progression., (© 2022 Wiley Periodicals LLC.)

Purpose: To develop a machine-learning algorithm and clinician-friendly tool predicting the likelihood of prolonged opioid use (>90 days) following hip arthroscopy., Methods: The Military Data Repository was queried for all adult patients undergoing arthroscopic hip surgery between 2012 and 2017. Demographic, health history, and prescription records were extracted for all included patients. Opioid use was divided into preoperative use (30-365 days before surgery), perioperative use (30 days before surgery through 14 days after surgery), postoperative use (14-90 days after surgery), and prolonged postoperative use (90-365 days after surgery). Six machine-learning algorithms (Naïve Bayes, Gradient Boosting Machine, Extreme Gradient Boosting, Random Forest, Elastic Net Regularization, and artificial neural network) were developed. Area under the receiver operating curve and Brier scores were calculated for each model. Decision curve analysis was applied to assess clinical utility. Local-Interpretable Model-Agnostic Explanations were used to demonstrate factor weights within the selected model., Results: A total of 6,760 patients were included, of whom 2,762 (40.9%) filled at least 1 opioid prescription >90 days after surgery. The artificial neural network model showed superior discrimination and calibration with area under the receiver operating curve = 0.71 (95% confidence interval 0.68-0.74) and Brier score = 0.21 (95% confidence interval 0.20-0.22). Postsurgical opioid use, age, and preoperative opioid use had the most influence on model outcome. Lesser factors included the presence of a psychological comorbidity and strong history of a substance use disorder., Conclusions: The artificial neural network model shows sufficient validity and discrimination for use in clinical practice. The 5 identified factors (age, preoperative opioid use, postoperative opioid use, presence of a mental health comorbidity, and presence of a preoperative substance use disorder) accurately predict the likelihood of prolonged opioid use following hip arthroscopy., Level of Evidence: III, retrospective comparative prognostic trial., (Published by Elsevier Inc.)

The Yorkshire-cross swine model is a valuable translational model commonly used to study cardiovascular physiology and response to insult. Although the effects of vasoactive medications have been well described in healthy swine, the effects of these medications during hemorrhagic shock are less studied. In this study, we sought to expand the utility of the swine model by characterizing the hemodynamic changes that occurred after the administration of commonly available vasoactive medications during euvolemic and hypovolemic states. To this end, we anesthetized and established femoral arterial, central venous, and pulmonary arterial access in 15 juvenile Yorkshire-cross pigs. The pigs then received a series of rapidly metabolized but highly vasoactive medications in a standard dosing sequence. After completion of this sequence, each pig underwent a 30-mL/kg hemorrhage over 10 min, and the standard dosing sequence was repeated. We then used standard sta- tistical techniques to compare the effects of these vasoactive medications on a variety of hemodynamic parameters between the euvolemic and hemorrhagic states. All subjects completed the study protocol. The responses in the hemorrhagic state were often attenuated or even opposite of those in the euvolemic state. For example, phenylephrine decreased the mean arterial blood pressure during the euvolemic state but increased it in the hemorrhagic state. These results clarify previously poorly defined responses to commonly used vasoactive agents during the hemorrhagic state in swine. Our findings also demonstrate the need to consider the complex and dynamic physiologic state of hemorrhage when anticipating the effects of vasoactive drugs and planning study protocols.

Intercellular signaling drives human development, but there is a paucity of in vitro models that recapitulate important tissue architecture while remaining operationally simple and scalable. As an example, formation of the upper lip and palate requires the orchestrated proliferation and fusion of embryonic facial growth centers and is dependent on paracrine epithelial-mesenchymal signaling through multiple pathways including the Sonic Hedgehog (SHH), transforming growth factor-beta (Tgf-β), bone morphogenic protein (BMP), and epidermal growth factor (EGF) pathways. We have developed a robust, throughput-compatible microphysiological system to model intercellular signaling including epithelial-mesenchymal interactions that is useful for studying both normal and abnormal orofacial development. We describe the construction and operation of an engineered microplate created using CNC micromilling of 96-well microtiter plates capable of containing up to 20 epithelial-mesenchymal microtissues. A dense three-dimensional mesenchyme is created by embedding cells (O9-1, 3T3) in a biomimetic hydrogel. An epithelial layer is then overlayed on the microtissue by loading cells in engineered microchannels that flank the microtissue. The result is an engineering epithelial-mesenchymal interface that is both on and perpendicular to the imaging plane making it suitable for high-content imaging and analysis. The resulting microtissues and device are compatible with diverse analytical techniques including fluorescent and luminescent cell health and enzymatic reporter assays, gene expression analyses, and protein staining. This tractable model and approach promise to shed light on critical processes in intercellular signaling events in orofacial development and beyond., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity in vitro is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable in vitro approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed in vivo . Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development., Competing Interests: BJ holds equity in Onexio Biosystems L.L.C. DB holds equity in Bellbrook Labs, L.L.C., Tasso, Inc., Stacks to the Future, L.L.C., Salus Discovery, L.L.C., Lynx Biosciences, Inc., and Onexio Biosystems L.L.C. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Johnson, Vitek, Morgan, Fink, Beames, Geiger, Beebe and Lipinski.)

Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution; however, this heterogeneity it is difficult to characterize. Here, we present a new approach-pathology-guided micropunching (PGM)-that enables identification and characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of live-cell tissue labeling using fluorescent antibodies/small molecules to identify heterogeneous foci (e.g., immune infiltrates or cells with high levels of reactive oxygen species) in viable tissues, coupled with a micropunch step to isolate cells from these heterogeneous foci for downstream molecular or vital functional analysis. Micropunches obtained from epithelial or stromal fibroblast foci in human prostate tissue show 6- to 12-fold enrichment in transcripts specific for EpCam/cytokeratin 8 and vimentin/a-smooth muscle actin/integrin 1-α, respectively. Transcriptional enrichment efficiency agrees with epithelial and stromal laser capture microdissection samples isolated from human prostate. Micropunched foci show a loss of cellular viability in the periphery, but centrally localized cells retained viability before and after dissociation and grew out in culture.

Objective: To determine the role of the aryl hydrocarbon receptor (AHR) in colitis-associated colorectal tumorigenesis., Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in United States. Chronic intestinal inflammation increases the risk for the development of CRC. We investigated the involvement of AHR, a ligand-activated transcriptional regulator, in colitis-associated colorectal tumorigenesis., Methods: We used a mouse model of colitis-associated colorectal tumorigenesis that employs treatment with azoxymethane and dextran sodium sulfate. We examined the role of AHR using both an Ahr-deletion mouse model (Ahr) and treatment with the AHR pro-agonist indole-3-carbinol (I3C). Incidence, multiplicity, and location of tumors were visually counted. Tumors were defined as neoplasms. Intestinal inflammation was assessed by quantitative PCR for proinflammatory markers and colon length. Data were evaluated and compared using GraphPad Prism software (version 6, La Jolla, CA)., Results: Tumor incidence was increased 32% in Ahr null mice and tumor multiplicity was approximately increased 3-fold compared with wild-type mice (2.4 vs 7; P < 0.05). Furthermore, tumor multiplicity was reduced 92% by treatment of I3C in wild-type mice, whereas the suppressor effect of I3C was not observed in Ahr null mice (P < 0.05)., Conclusions: We found that AHR plays a protective role in colitis-associated colorectal tumorigenesis. This conclusion is based on the observations that Ahr null mice showed increased number of colorectal tumors, and mice treated with I3C exhibited fewer tumors. This study supports the use of AHR agonists such as I3C as a chemopreventive therapy for IBD-associated CRC in human patients., Competing Interests: All authors have no conflict of interest.

The polyphenolic flavone chrysin has been evaluated as a natural chemopreventive agent due to its anti-cancer effects in a variety of cancer cell lines. However, the mechanism of the chemopreventive effect has been not well established, especially in human colorectal cancer cells. We evaluated the chemopreventive effect of chrysin in three different human colorectal cancer cell lines. We found that chrysin treatment consequently reduced cell viability via induction of apoptosis. We identified that the involvement of up-regulation of pro-apoptotic cytokines tumor necrosis factor (Tnf) α and β genes and consequent activation of the TNF-mediated transcriptional pathway in chrysin-induced apoptosis. Using our generated AHR siRNA expressing colorectal cancer cells, we demonstrated that the chrysin-induced up-regulation of Tnfα and β gene expression was dependent on the aryl hydrocarbon receptor (AHR), which is a ligand-receptor for chrysin. Subsequently, we found that the AHR siRNA expressing colorectal cancer cells were resistant to chrysin-induced apoptosis. Therefore, we concluded that AHR is required for the chrysin-induced apoptosis and the up-regulation of Tnfα and β gene expression in human colorectal cancer cells., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: In early-stage rectal cancer, the surgeon must decide between the high morbidity of radical surgery and the high recurrence rates of local excision. A prognostic marker could improve patient selection and lower recurrence rates. Micro-ribonucleic acids (miRNAs), small RNAs that often inhibit tumor suppressors, have shown prognostic potential in colorectal cancer. We hypothesized that high miRNA levels in malignant tissue from early-stage rectal cancer patients could predict recurrence after local excision., Materials and Methods: We identified 17 early-stage rectal cancer patients treated with local excision between 1990 and 2005, four of whom had recurrences. Total RNA was extracted from benign and malignant tissue and used in quantitative real-time reverse transcriptase polymerase chain reaction to probe for miR-20a, miR-21, miR-106a, miR-181b, and miR-203. MiRNA data were evaluated for association with recurrence using univariate analysis with Wilcoxon rank sum test., Results: Malignant tissue in both patients who had recurrences and patients who did not have recurrences had equivalently high levels of miRNA. However, the benign tissue of patients who recurred contained significantly higher levels of all five miRNAs when compared with the benign tissue of nonrecurrent patients despite having no histological differences., Conclusions: This is the first study to show that high miRNA levels of histologically benign tissue obtained from the surgical margin of locally excised rectal cancers can predict recurrence. The malignant miRNA levels did not have predictive value. Further investigation of miRNAs is needed to explore their potential for a more accurate prognosis of rectal cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The metal chelator Triapine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, is a potent inhibitor of ribonucleotide reductase. EPR spectra consistent with signals from Fe-transferrin, heme, and low-spin iron or cupric ion were observed in peripheral blood mononuclear cells (PBMCs) obtained from patients treated with Triapine. One signal that is unequivocally identified is the signal for Fe-transferrin. It is hypothesized that Fe uptake is blocked by reactive oxygen species generated by FeT(2) or CuT that damage transferrin or transferrin receptor. A potential source for the increase in the heme signal is cytochrome c released from the mitochondria. These results provide valuable insight into the in vivo mechanism of action of Triapine.

Photodynamic therapy with 5-aminolevulinic acid (ALA) is based on metabolism of ALA to a photosensitizing agent, protoporphyrin IX (PpIX), in tumor cells. Photosensitivity of target cells may be influenced by mitochondrial iron levels because ferrochelatase-catalyzed insertion of Fe2+ into PpIX converts it to heme, a nonsensitizer. To investigate this prospect, we exposed L1210 cells (approximately 10(6)/mL in 1% serum-containing medium) to a lipophilic iron chelate, ferric-8-hydroxyquinoline (Fe[HQ]2, 0.5 microM), prior to treating with ALA (0.2 mM, 4 h) and irradiating with broadband visible light. When Fe(HQ)2 was added to cells immediately or 1 h before ALA, the initial rate of photokilling, as measured by thiazolyl blue (mitochondrial dehydrogenase) assay, was markedly less than that of non-iron controls. The HPLC analysis of cell extracts indicated that ALA-induced PpIX was at least 50% lower after this Fe(HQ)2 treatment, presumably explaining the drop in photolethality. By contrast, cells treated with ALA and light 20 h after being exposed to Fe(HQ)2 contained the same amount of PpIX as non-iron controls and were photoinactivated at nearly the same rate. The 20 h delayed cells contained approximately 12 times more immunodetectable ferritin heavy subunit than controls or 1 h counterparts, which could account for the disappearance of iron's antisensitization effects in the former. Consistent with this idea, the short-term effects of Fe(HQ)2 on ALA-induced sensitization were found to be blunted significantly in ferritin-enriched cells. The Fe(HQ)2 produced strikingly different results when cells were sensitized with exogenous PpIX, stimulating photokilling after short-term contact but inhibiting it after long-term contact while having no significant effect on the level of cell-associated PpIX in either case. Thus, iron can have diverse effects on PpIX-mediated photokilling, depending on contact time with cells and whether the porphyrin is metabolically derived or applied as such.

In addition to the applications described, HPLC-EC(Hg) can be used for determining LOOHs in lipoproteins and for monitoring LOOH detoxification in cells. As it continues to be developed and refined, this approach should prove to be valuable not only for ultrasensitive determination of lipid-derived peroxides, but protein- and nucleic acid-derived peroxided as well.

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemoglobin, is a potential source of prooxidant iron in heavily vascularized tumors. We have evaluated hemin's effects on photodynamic inactivation of bovine artery endothelial cells, using a partially purified oligomeric fraction of hematoporphyrin derivative (HPD-A) as the sensitizing agent. Confluent cells in 5% serum/RPMI medium showed a progressive loss of thiazolyl blue (MTT)-detectable viability when irradiated with broadband visible light in the presence of HPD-A. Cells pretreated with desferrioxamine (DFO) were substantially less sensitive to photokilling, implying that non-heme iron plays a role in cytotoxic activity. Hemin (10-20 microM) had remarkably different effects on photokilling, depending on the time interval between adding it to cells and exposing them to photodynamic action. For example, cells were more sensitive when photostressed immediately after 1 h hemin treatment and washing but much more resistant when photostressed 23 h later. Similar responses were observed when cells were challenged with glucose oxidase. Immunoblot analysis following hemin treatment revealed a progressive induction of the heavy (H) subunit of ferritin that paralleled the development of hyperresistance. After incubation with saturating levels of the synthetic iron donor [55Fe]ferric-8-hydroxyquinoline, hemin-stimulated cells contained about four times more immunoprecipitable ferritin 55Fe than controls. This is consistent with the notion that sequestration of toxic iron as a result of induction of H-chain-enriched ferritin is a key factor in hyperresistance. Inflammatory injury in tumor vasculatures could expose endothelial and neoplastic cells to chronic hemoglobin-derived iron. Consequent upregulation of ferritin could impact negatively on the efficacy of photodynamic therapy and other oxidant-based cancer therapies.

Photodynamic action of merocyanine 540, an antileukemic sensitizing dye, on murine L1210 cells results in the formation of lipid hydroperoxides and loss of cell viability. High-performance liquid chromatography with mercury cathode electrochemical detection was used for determining lipid oxidation products, including the following cholesterol-derived hydroperoxides: 5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, and unresolved 7 alpha, 7 beta-OOH. Among these species, 5 alpha-, 6 alpha-, and 6 beta-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas 7 alpha- and 7 beta-OOH (products of free radical reactions) became so after prolonged irradiation or during dark incubation after exposure to a light dose. These mechanistic changes were studied in a unique way by monitoring shifts in the peroxide ratio, i.e., 7-OOH/5 alpha-OOH, or 7-OOH/6-OOH. When cells (10(7)/ml) were exposed to a visible light fluence of 0.6 J/cm2 in the presence of 10 microM merocyanine 540, 7-OOH/5 alpha-OOH increased by approximately 100% after 2 h of dark incubation at 37 degrees C. The increase was much larger (approximately 250%) when cells were photooxidized after treatment with 1 microM ferric-8-hydroxyquinoline, a lipophilic iron donor, whereas no increase was observed when cells were pretreated with 100 microM desferrioxamine, an avid iron chelator/redox inhibitor. Correspondingly, postirradiation formation of thiobarbituric acid-reactive material was markedly enhanced by ferric-8-hydroxyquinoline and suppressed by desferrioxamine, as was the extent of cell killing. When added to cells after a light dose, chain-breaking antioxidants such as butylated hydroxytoluene and alpha-tocopherol strongly protected against cell killing and slowed the increase in 7-OOH/5 alpha-OOH ratio. It is apparent from these results that (1) the 7-OOH/5 alpha-OOH or 7-OOH/6-OOH ratio can be used as a highly sensitive index of singlet oxygen vs. free radical dominance in photodynamically stressed cells; and (2) that postirradiation chain peroxidation plays an important role in photodynamically initiated cell killing.

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is a selenoenzyme that can catalyze the direct reduction of various membrane lipid hydroperoxides and by so doing could play a vital role in cytoprotection against peroxidative damage. The activity of purified testicular PHGPX on several photochemically-generated cholesterol hydroperoxide (ChOOH) species was investigated, using high-performance liquid chromatography with electrochemical detection for peroxide analysis and thinlayer chromatography with 14C-radiodetection for diol product analysis. The following ChOOH isomers were monitored: 5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH (singlet oxygen adducts), and unresolved 7 alpha,7 beta-OOH (derived from 5 alpha-OOH rearrangement). Apparent first-order rate constants for GSH/PHGPX-induced peroxide loss (or diol accumulation) in Triton X-100 micelles, unilamellar liposomes, or erythrocyte ghost membranes increased in the following order: 5 alpha-OOH < 6 alpha-OOH approximately equal to 7 alpha,7 beta-OOH < 6beta-OOH. A similar trend was observed when the peroxides were incubated with Triton Iysates of Se-replete L1210 or K562 cells, implicating PHGPX in these reactions. Consistent with this, there was little or no ChOOH reduction if GSH was omitted or if lysates from Se-deprived cells were used. Liposomal 5 alpha-OOH was found to be much more cytotoxic than equimolar liposomal 6 beta-OOH, producing a 50% loss of L1210 clonogenicity at approximately 1/5 the concentration of the latter. Faster uptake of 5 alpha-OOH was ruled out as the basis for greater cytotoxicity, suggesting that relatively inefficient metabolism by the GSH/PHGPX system might be the reason. As supporting evidence, it was found that cells accumulate the diol reduction product of 5 alpha-OOH more slowly than that of 6 beta-OOH during incubation with the respective peroxides. Slow detoxification coupled with rapid formation makes 5 alpha-OOH potentially the most damaging ChOOH to arise in cells exposed to singlet oxygen.

Singlet oxygen (1O2)-mediated photooxidation of cholesterol gives three hydroperoxide products: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH) and 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH). These species have been compared with respect to photogeneration rate on the one hand and susceptibility to enzymatic reduction/detoxification on the other, using the erythrocyte ghost as a cholesterol-containing test membrane and chloroaluminum phthalocyanine tetrasulfonate (AlPcS4) as a 1O2 sensitizer. Peroxide analysis was accomplished by high-performance liquid chromatography with mercury cathode electrochemical detection (HPLC-EC[Hg]). The initial rate of 5 alpha-OOH accumulation in AlPcS4/light-treated ghosts was found to be about three times greater than that of 6 alpha-OOH or 6 beta-OOH. Membranes irradiated in the presence of ascorbate and ferric-8-hydroxyquinoline (Fe[HQ]2, a lipophilic iron complex) accumulated lesser amounts of 5 alpha-OOH, 6 alpha-OOH and 6 beta-OOH but relatively large amounts of another peroxide pair, 3 beta-hydroxycholest-5-ene-7 alpha- and 7 beta-hydroperoxide (7 alpha, 7 beta-OOH), suggestive of iron-mediated free radical peroxidation. When photoperoxidized membranes containing 5 alpha-OOH, 6 alpha,6 beta-OOH and 7 alpha,7 beta-OOH (arising from 5 alpha-OOH rearrangement) were incubated with glutathione (GSH) and phospholipid hydroperoxide glutathione peroxidase (PHGPX), all hydroperoxide species underwent HPLC-EC(Hg)-detectable reduction to alcohols, the relative first order rate constants being as follows: 1.0 (5 alpha-OOH), 2.0 (7 alpha,7 beta-OOH), 2.4 (6 alpha-OOH) and 3.2 (6 beta-OOH).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were < 0.3 pmol and < 30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.

Murine leukemia L1210 cells rendered deficient in glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) by Se deprivation (L.Se(-) cells) were found to be more sensitive to tert-butyl hydroperoxide (t-BuOOH) cytotoxicity than Se-replete controls (L.Se(+) cells). Human K562 cells, which express PHGPX, but not GPX, were also more sensitive to t-BuOOH in the Se-deficient (K.Se(-)) than Se-satisfied (K.Se(+)) condition. In examining the metabolic basis for selenoperoxidase-dependent resistance, we found that glucose-replete Se(-) cells reduce t-BuOOH to t-butanol far more slowly than Se(+) cells, the ratio of the first-order rate constants approximating that of the GPX activities (L1210 cells) or PHGPX activities (K562 cells). Monitoring peroxide-induced changes in GSH and GSSG gave consistent results; e.g., glucose-depleted L.Se(+) cells exhibited a first order loss of GSH that was substantially faster than that of glucose-depleted L.Se(-) cells. Under the conditions used, peroxide-induced conversion of GSH to GSSG could be stoichiometrically reversed by resupplying D-glucose, indicating that no significant lysis or GSSG efflux and/or interchange had taken place. The apparent first-order rate constant for GSH decay increased progressively for L1210 cells expressing a range of GPX activities from approximately 5% to 100%, demonstrating that peroxide detoxification is strictly dependent on enzyme content. The initial rate of 14CO2 release from D-[1-14C]glucose supplied in the medium was much greater for L.Se(+) or K.Se(+) cells than for their respective Se(-) counterparts, consistent with greater hexose monophosphate shunt activity in the former. These results highlight the importance of selenoperoxidase action in the glutathione cycle as a means by which tumor cells cope with hydroperoxide stress.

Murine leukemia L1210 cells grown for 5-7 d in the presence of 1% serum without added selenium [Se(-) cells] expressed < 5% of the glutathione peroxidase (GPX) activity of selenium-supplemented controls [Se(+) cells]. Clonogenic survival assays indicated that t-butyl hydroperoxide (t-BuOOH) is much more toxic to Se(-) cells (LC50 approximately 10 microM) than to Se(+) or selenium-repleted [Se(-/+)] cells (LC50 approximately 250 microM). Hypersensitivity of Se(-) cells to t-BuOOH was partially reversed by treating them with Ebselen, a selenoperoxidase mimetic; thus, selenoperoxidase insufficiency was probably the most serious defect of Se deprivation. Cytotoxicity of t-BuOOH was inhibited by desferrioxamine and by alpha-tocopherol, indicating that redox iron and free radical intermediates are involved. Elevated sensitivity of Se(-) cells to t-BuOOH was accompanied by an increased susceptibility to free radical lipid peroxidation, which became even more pronounced in cells that had been grown in arachidonate (20:4, n-6) supplemented media. That glutathione (GSH) is required for cytoprotection was established by showing that Se(+) cells are less resistant to t-BuOOH after exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. Coupled enzymatic assays indicated that Se(+) or Se(-/+) cells metabolize t-BuOOH 20-25 times more rapidly than Se(-), consistent with the measured difference in GPX activities of these cells. Correspondingly, when challenged with t-BuOOH, Se(+) cells showed an initial loss of GSH and elevation of GSSG that exceeded that of Se(-) cells. It was further shown that like Se(-) cells, BSO- or BCNU-treated Se(+) cells metabolize t-BuOOH more slowly than nontreated controls. These results clearly indicate that selenoperoxidase action in the glutathione cycle is a vital element in cellular defense against toxic hydroperoxides.

Photodynamic therapy with the lipophilic sensitizing dye merocyanine 540 (MC540) is a promising new approach for extracorporeal purging of neoplastic cells from autologous remission bone marrow grafts. Resistance-conferring cellular defenses against the cytotoxic effects of MC540/photodynamic therapy have not been well characterized. This study focuses on the cytoprotective effects of the glutathione-dependent selenoperoxidases GPX and PHGPX, which can detoxify a wide variety of hydroperoxides, including lipid-derived species (LOOHs). Murine leukemia L1210 cells were grown in 1% serum media without [L.Se(-)] and with [L.Se(+)] selenium supplementation. L.Se(-) cells expressed 10- to 20-fold lower GPX and PHGPX activities than L.Se(+) controls and were markedly more sensitive to MC540-mediated photoperoxidation (LOOH formation) and clonally assessed photokilling. Susceptibility of L.Se(-) cells to photoperoxidation and photokilling could be fully reversed to L.Se(+) levels by replenishing Se, and partially reversed by treating with Ebselen, a selenoperoxidase mimetic. Altered lipid composition, greater uptake of MC540, and defective catabolism of H2O2 were all ruled out as possible factors in the elevated photosensitivity of L.Se(-) cells. Human leukemia K562 cells (capable of expressing PHGPX but not GPX) exhibited 5- to 10-fold lower PHGPX activity under Se-deficient relative to Se-sufficient conditions. Although MC540 uptake (nmol/mg lipid) by K562 and L1210 cells was essentially the same, the former were more resistant to photoinactivation. However, like murine counterparts, Se-deficient cells were more susceptible to photoperoxidation and photokilling than Se-sufficient controls. These results clearly demonstrate that GPX and/or PHGPX in L1210 cells and PHGPX in K562 cells play an important cytoprotective role during photooxidative stress. Whether membrane damage due to lipid photoperoxidation is causally related to cell death is not certain; however, the parallel effects of Se deficiency on LOOH formation and cell killing are at least consistent with this possibility.

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.

Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxidized liposomes underwent a burst of thiobarbituric acid-detectable lipid peroxidation which could be inhibited by prior GSH/PHGPX treatment, but not by GSH/GPX treatment. Additional experiments indicated that hydroperoxides of phosphatidylcholine, cholesterol and cholesteryl esters in low-density lipoprotein are also good substrates for PHGPX. An important role of PHGPX in cellular detoxification of a wide variety of LOOHs in membranes and internalized lipoproteins is suggested from these findings.