Proliferation-attenuating and apoptosis-inducing effects of tryptanthrin on human chronic myeloid leukemia k562 cell line in vitro

Tryptanthrin, a kind of indole quinazoline alkaloid, has been shown to exhibit anti-microbial, anti-inflammation and anti-tumor effects both in vivo and in vitro. However, its biological activity on human chronic myeloid leukemia cell line K562 is not fully understood. In the present study, we investigated the proliferation-attenuating and apoptosis-inducing effects of tryptanthrin on leukemia K562 cells in vitro and explored the underlying mechanisms. The results showed that tryptanthrin could significantly inhibit K562 cells proliferation in a time- and dose-dependent manner as evidenced by MTT assay and flow cytometry analysis. We also observed pyknosis, chromatin margination and the formation of apoptotic bodies in the presence of tryptanthrin under the electron microscope. Nuclei fragmentation and condensation by Hoechst 33258 staining were detected as well. The amount of apoptotic cells significantly increased whereas the mitochondrial membrane potential decreased dramatically after tryptanthrin exposure. K562 cells in the tryptanthrin treated group exhibited an increase in cytosol cyt-c, Bax and activated caspase-3 expression while a decrease in Bcl-2, mito cyt-c and pro-caspase-3 contents. However, the changes of pro-caspase-3 and activated caspase-3 could be abolished by a pan-caspase inhibitor ZVAD-FMK. These results suggest that tryptanthrin has proliferation-attenuating and apoptosis-inducing effects on K562 cells. The underlying mechanism is probably attributed to the reduction in mitochondria membrane potential, the release of mito cyt-c and pro-caspase-3 activation.     

多烯紫杉醇诱导细胞周期阻断与凋亡过程的模型

以多烯紫杉醇诱导白血病细胞株K562为对象,建立了描述肿瘤细胞生长及其与药物作用关系的细胞周期数学模型,研究了多烯紫杉醇诱导K562细胞周期变化与凋亡现象及量效关系.结果表明,多烯紫杉醇引起K562细胞M期阻断和诱导细胞凋亡的饱和浓度分别为17.96、7.82 nmol·L-1,有效浓度分别为2.63、1.69 nmol·L^-1;低浓度（1.69～2.63 nmol·L^-1）的多烯紫杉醇直接诱导细胞凋亡而不引起明显的M期阻断;高浓度（>7.82 nmol·L^-1）的多烯紫杉醇主要效应是促进细胞M期阻断.本模型揭示出M期阻断与凋亡无显著的相关性,为多烯紫杉醇的作用机制提供了一个新的解释.     

透明质酸对人骨性关节炎软骨细胞凋亡及细胞周期的影响

目的观察透明质酸(Hyaluronic acid,HA)对人骨性关节炎(Osteoarthritis,OA)软骨细胞凋亡及细胞周期的影响,以探讨HA保护软骨细胞的作用机制。方法分离人正常软骨细胞和OA软骨细胞,传至第2代后,分别经HA处理24 h,采用MTT法检测细胞的增殖活力,流式细胞术检测细胞凋亡率和细胞周期。结果 HA能明显降低人OA软骨细胞的凋亡率(P<0.05),提高细胞的增殖活力、S期比例和增殖指数(P<0.05),且对正常人软骨细胞的增殖活力和凋亡无明显影响。结论 HA可促进OA软骨细胞的分裂与增殖,降低细胞凋亡率,从而对软骨细胞发挥保护作用。     

狂犬病毒在Vero细胞上的传代适应

本文研究了3株狂犬病毒(C_(42)、C_(103)及4aG株)在Vero细胞上的培养条件和方法,并通过连续传代培养,选出1株滴度平均达到7.13LogLD_(50)/ml以上,达到WHO规定的不需浓缩的标准(≥10~(7.0)ICLD_(50)/ml)毒株—C_(42)—V_(170)病毒的增殖高峰由原来的10天左右提前到3～5天。另外2株的病毒滴度也达到了WHO规定的制备人用Vero细胞狂犬疫苗(PVRV)的标准(≥10~(6.0)ICLD_(50)/ml)。3株病毒都能在Vero细胞上产生蚀斑,符合WHO提出的疫苗生产毒种的要求。     

RNA干扰Apg-2基因表达对BaF3-MIGR1和BaF3-p210细胞凋亡的影响

目的探讨RNA干扰Apg-2基因表达对BaF3-MIGR1和BaF3-p210(Bp210)细胞凋亡的影响。方法取BaF3-MIGR1和Bp210、稳定抑制Apg-2表达的BaF3-MIGR1-Hspa42和Bp210-Hspa42细胞及阴性对照Bp210-HspaHK和BaF3-MIGR1-HspaHK细胞,采用瑞特染色法观察各组细胞的形态学变化;流式细胞术检测各组细胞的凋亡情况;Western blot法检测各组细胞中凋亡相关蛋白Bax和Bcl-2的表达水平。结果干扰Apg-2基因表达后,细胞出现肿胀、胞浆空泡、核染色质聚集、核碎裂,部分细胞可见凋亡小体,处于有丝分裂中期细胞明显减少。与BaF3-MIGR1和Bp210细胞相比,BaF3-MIGR1-Hspa42和Bp210-Hspa42细胞的凋亡率显著增加(P<0.05);Bcl-2蛋白的表达水平下调,Bax蛋白的表达水平无明显变化。结论干扰Apg-2基因表达可促进BaF3-MIGR1和Bp210细胞凋亡增加,其可能是通过降低抗凋亡蛋白Bcl-2的表达水平来实现的。     

Changes of constituents and activity to apoptosis and cell cycle during fermentation of tea

Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest.     

Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4β,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC₅₀ values in the range 3.6–13.5 μM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1–3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258–propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.     

Study of the anticancer properties of tin(IV) carboxylate complexes on a panel of human tumor cell lines

A group of organotin(IV) complexes were prepared: [SnCy₃(DMNI)] ( 1 ), [SnCy₃(BZDO)] ( 2 ), [SnCy₃(DMFU)] ( 3 ), and [SnPh₂(BZDO)₂] ( 4 ), for which DMNIH=2,6‐dimethoxynicotinic acid, BZDOH=1,4‐benzodioxane‐6‐carboxylic acid, and DMFUH=2,5‐dimethyl‐3‐furoic acid. The cytotoxic activities of compounds 1 – 4 were tested against pancreatic carcinoma (PANC‐1), erythroleukemia (K562), and two glioblastoma multiform (U87 and LN‐229) human cell lines; they show very high antiproliferative activity, with IC₅₀ values in the 150–700 nM range after incubation for 72 h. Distribution of cellular DNA upon treatment with 1 – 4 revealed that whereas compounds 1 – 3 induce apoptosis in most of the cell lines, compound 4 does not affect cell viability in any cell line tested, indicating a possible difference in cytotoxic mechanism. Studies with the daunomycin‐resistant K562/R cell line expressing P‐glycoprotein (Pgp) showed that compounds 1 – 4 are not substrates of this protein efflux pump, indicating that these compounds do not induce acquisition of multidrug resistance, which is associated with the overexpression of Pgp.     

GINS2基因siRNA真核表达质粒的构建及其对NB4细胞凋亡的影响

目的构建GINS2基因siRNA真核表达质粒,并检测其对NB4细胞凋亡的影响。方法人工合成针对GINS2基因的4组siRNA干扰序列和1组无同源性的序列,测序鉴定后转染低传代人早幼粒细胞系NB4细胞,经G418筛选后,通过QRT-PCR、Western blot检测重组质粒对NB4细胞中GINS2基因mRNA转录水平和蛋白表达水平的影响;流式细胞术检测NB4细胞的凋亡情况。结果 5组重组质粒经测序证明构建正确,4组干扰质粒转染NB4细胞后,细胞中GINS2基因mRNA的转录水平和蛋白的表达水平均有所降低,其中干扰组1基因干扰率达50%,蛋白相对表达量为56%;干扰组1细胞凋亡率为32.54%,较正常对照组和NC组明显上升(P<0.01)。结论成功构建了GINS2基因siRNA真核表达质粒,抑制GINS2基因的表达可促进NB4细胞的凋亡,为进一步研究GINS2基因在白血病中的作用奠定了基础。     

乙型肝炎病毒X蛋白对小鼠胚胎肝干细胞凋亡及相关蛋白表达的影响

目的研究乙型肝炎病毒(hepatitis B virus,HBV)X蛋白(HBx)对小鼠胚胎肝干细胞(embryonic liver stemcell,ELSC)凋亡及相关蛋白Bcl2、Mcl1、Bax表达的影响。方法采用表达绿色荧光蛋白(green fluorescent protein,GFP)的腺病毒载体系统将HBx基因转入小鼠胚胎肝干细胞ELSC14.5中,采用RT-PCR和Western blot法检测细胞中HBx基因mRNA的转录和蛋白的表达;Hoechst33342染色法观察细胞核的改变;TUNEL法和流式细胞术检测细胞的凋亡情况;Real-time PCR和Western blot法检测抗凋亡因子Bcl2、Mcl1和促凋亡因子Bax基因mRNA的转录水平和蛋白的表达水平。结果重组腺病毒Ad-GFP-HBx能有效感染ELSC14.5细胞,HBx基因和蛋白均能特异性表达;感染的ELSC14.5细胞核呈现固缩,且边缘化的细胞数减少,细胞凋亡率降低;细胞中Bcl2和Mcl1基因mRNA的转录水平和蛋白的表达水平均增高,而Bax的表达降低。结论 HBx可通过调节Bcl2家族中抗凋亡因子和促凋亡因子的比例失衡,来抑制小鼠胚胎肝干细胞的凋亡,促进其存活。     

Inhibition of Bcr-Abl phosphorylation and induction of apoptosis by pyrazolo[3,4-d]pyrimidines in human leukemia cells

A series of pyrazolo[3,4-d]pyrimidines, previously found to be Src inhibitors, was tested for their ability to inhibit proliferation of three Bcr-Abl-positive human leukemia cell lines (K-562, KU-812, and MEG-01), on the basis of the experimental evidence that various Src inhibitors are also active against Bcr-Abl kinase (the so called dual Src/Abl inhibitors). They reduce Bcr-Abl tyrosine phosphorylation and promote apoptosis of the Bcr-Abl-expressing cells. A cell-free enzymatic assay on isolated c-Abl confirmed that such compounds directly inhibit Abl activity. Finally, molecular modeling simulations were also performed to hypothesize the binding mode of the compounds into the Abl binding site.     

Cytotoxic salan-titanium(IV) complexes: high activity toward a range of sensitive and drug-resistant cell lines, and mechanistic insights

The cytotoxicities of highly efficient salan-Ti(IV) complexes toward a range of cell lines, including drug-resistant cells, are reported along with preliminary mechanistic insights. Five salan-Ti(IV) complexes were investigated toward eight different human and murine cancer-derived cell lines, including colon, ovarian, lung, cervical, pancreatic, leukemic, skin, and breast. The salan complexes are more active toward the cells analyzed than cisplatin and the known titanium compound (bzac)(2) Ti(OiPr)(2) , and no cell line resistant to the salan complexes was identified. Moreover, the salan-Ti(IV) complexes are highly active toward both cisplatin-sensitive (A2780) and cisplatin-resistant (A2780CisR) human ovarian cancer cell lines. Similarly, the salan complexes are cytotoxic toward multi-drug-resistant (ABCB1-expressing) mouse lymphoma cell lines HU-1 and HU-2. Importantly, minimal or no activity was observed toward primary murine cells (bone marrow, heart, liver, kidney, spleen, and lung), supporting selectivity for cancer cells. Additionally, the salan complexes maintain high cytotoxicity for up to 24 h following exposure to cell culture medium, whereas reference complexes (bzac)(2) Ti(OiPr)(2) and Cp(2) TiCl(2) rapidly lose much of their activity upon exposure to medium, within ~1 h. The upregulation of p53 followed by cell-cycle arrest in G(1) phase is likely one mechanism of action of the salan complexes. Taken together, the results indicate that these compounds are selectively toxic to cancer cells and are able to circumvent two independent mechanisms of drug resistance, thus expanding the scope of their potential medicinal utility.     

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.     

Synthesis of a novel series of 2-methylsulfanyl fatty acids and their toxicity on the human K-562 and U-937 leukemia cell lines

The hitherto unknown 2-methylsulfanyldecanoic acid and 2-methylsulfanyldodecanoic acid were synthesized from methyl decanoate and methyl dodecanoate, respectively, through the reaction of lithium diisopropylamide and dimethyldisulfide in THF followed by saponification with potassium hydroxide in ethanol. Both α-methylsulfanylated FA were cytotoxic to the human chronic myelogenous leukemia K-562 and the human histiocytic lymphoma U-937 cell lines with EC₅₀ values in the 200–300 μM range, which makes them more cytotoxic to these cell lines than decanoic and/or dodecanoic acid. The cytotoxicity of the studied FA toward K-562 followed the order 2-SCH₃-12∶0>2-SCH₃-10∶0>10∶0>12∶0>2-OCH₃-12∶0, whereas toward U-937 the cytotoxicity was 2-SCH₃-10∶0>2-SCH₃-12∶0>12∶0>10∶0>2-OCH₃-12∶0. These results indicate that the α-methylsulfanyl substitution increases the cytotoxicity of the C₁₀ and C₁₂ FA toward the studied leukemia cell lines.     

Cytotoxicity of Newly Synthesized Quinazoline–Sulfonamide Derivatives in Human Leukemia Cell Lines and Their Effect on Hematopoiesis in Zebrafish Embryos

Many quinazoline derivatives with pharmacological properties, such as anticancer activity, have been synthesized. Fourteen quinazoline derivatives bearing a substituted sulfonamide moiety (4a–n) were previously synthesized and fully characterized. These compounds exerted antiproliferative activity against cell lines derived from solid tumors. Herein, the antileukemic activities of these compounds (4a–n) against two different leukemia cell lines (Jurkat acute T cell and THP-1 acute monocytic) were investigated. Our investigation included examining their activity in vivo in a zebrafish embryo model. Remarkably, compounds 4a and 4d were the most potent in suppressing cell proliferation, with an IC₅₀ value range of 4–6.5 µM. Flow cytometry analysis indicated that both compounds halted cell progression at the G2/M phase and induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analyses also showed that both compounds effectively induced apoptosis by upregulating the expression of proapoptotic factors while downregulating that of antiapoptotic factors. In vivo animal toxicity assays performed in zebrafish embryos indicated that compound 4d was more toxic than compound 4a, with compound 4d inducing multiple levels of teratogenic phenotypes in zebrafish embryos at a sublethal concentration. Moreover, both compounds perturbed the hematopoiesis process in developing zebrafish embryos. Collectively, our data suggest that compounds 4a and 4d have the potential to be used as antileukemic agents.     

Effects of primer and annealing treatments on the shear strength between anodized Ti6Al4V and epoxy

The effects of primer and annealing treatments on the shear strength between anodized Ti6Al4V and epoxy were investigated. Primer coating improved the shear strength between anodized Ti alloy and epoxy by up to 81.3% using concurrent curing compared with that of control specimens. After annealing of anodized Ti alloy and applying primer, the shear strength of the specimen was further increased by 6.4% due to the formation of stable TiO₂ transferred from TiO in the anodization process. SEM analysis revealed the specimen without primer and annealing treatments showed adhesive failure between epoxy–alloy interface and discontinuous cohesive failure of epoxy. Primer coating initiated a new interfacial failure mode between the oxide layer and alloy due to the improved bonding strength between epoxy and oxide layer. In addition, annealing and primer treatments generated large tracts of epoxy continuous cohesive failure, showing good agreement with its higher shear strength and work of fracture.     

The Enhanced Cytotoxic Effects in B-Cell Leukemia and Lymphoma Following Activation of Prostaglandin EP4 Receptor and Targeting of CD20 Antigen by Monoclonal Antibodies

Anti-CD20 monoclonal antibodies (MAbs) have revolutionized the treatment of B-cell leukemia and lymphoma. However, many patients do not respond to such treatment due to either deficiency of the complementary immune response or resistance to apoptosis. Other currently available treatments are often inadequate or induce major side effects. Therefore, there is a constant need for improved therapies. The prostaglandin E2 receptor 4 (EP4) receptor has been identified as a promising therapeutic target for hematologic B-cell malignancies. Herein, we report that EP4 receptor agonists PgE1-OH and L-902688 have exhibited enhanced cytotoxicity when applied together with anti-CD20 MAbs rituximab, ofatumumab and obinutuzumab in vitro in Burkitt lymphoma cells Ramos, as well as in p53-deficient chronic lymphocytic leukemia (CLL) cells MEC-1. Moreover, the enhanced cytotoxic effects of EP4 receptor agonists and MAbs targeting CD20 have been identified ex vivo on primary lymphocytes B obtained from patients diagnosed with CLL. Incubation of cells with PgE1-OH and L-902688 preserved the expression of CD20 molecules, further confirming the anti-leukemic potential of EP4 receptor agonists in combination with anti-CD20 MAbs. Additionally, we demonstrated that the EP4 receptor agonist PgE-1-OH induced apoptosis and inhibited proliferation via the EP4 receptor triggering in CLL. This work has revealed very important findings leading towards the elucidation of the anticancer potential of PgE1-OH and L-902688, either alone or in combination with MAbs. This may contribute to the development of potential therapeutic alternatives for patients with B-cell malignancies.     

Synthesis and antitumor molecular mechanism of agents based on amino 2-(3',4',5'-trimethoxybenzoyl)benzo[b]furan: inhibition of tubulin and induction of apoptosis

Induction of apoptosis is a promising strategy that could lead to the discovery of new molecules active in cancer chemotherapy. This property is generally observed when cells are treated with agents that target microtubules, dynamic structures that play a crucial role in cell division. Small molecules such as benzo[b]furans are attractive as inhibitors of tubulin polymerization. A new class of inhibitors of tubulin polymerization based on the 2-(3',4',5'-trimethoxybenzoyl)benzo[b]furan molecular skeleton, with the amino group placed at different positions on the benzene ring, were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell-cycle effects. The methoxy substitution pattern on the benzene portion of the benzo[b]furan moiety played an important role in affecting antiproliferative activity. In the series of 5-amino derivatives, the greatest inhibition of cell growth occurred if the methoxy substituent is placed at the C6 position, whereas C7 substitution decreases potency. The most promising compound in this series is 2-(3',4',5'-trimethoxybenzoyl)-3-methyl-5-amino-6-methoxybenzo[b]furan (3 h), which inhibits cancer cell growth at nanomolar concentrations (IC(50) =16-24 nM), and interacts strongly with tubulin by binding to the colchicine site. Sub-G(1) apoptotic cells in cultures of HL-60 and U937 cells were observed by flow cytometric analysis after treatment with 3 h in a concentration-dependent manner. We also show that compound 3 h induces apoptosis by activation of caspase-3, -8, and -9, and this is associated with cytochrome c release from mitochondria. The introduction of an α-bromoacryloyl group increased antiproliferative activity with respect to the parent amino derivatives.     

Effects of polyunsaturated fatty acids and their n-6 hydroperoxides on growth of five malignant cell lines and the significance of culture media

We examined effects of polyunsaturated fatty acids (PUFA), their corresponding hydroperoxy fatty acids (hp-PUFA), as well as various pro- and antioxidants on the growth of tumor cells in culture. When cultured in RPMI 1640 medium, A-427 and WEHI clone 13 cells were both highly sensitive to hydroperoxy docosahexaenoic acid (hp-DHA), but they were far less sensitive in minimum essential medium (MEM). In contrast, A-427 cells were also sensitive to DHA in both culture media, while WEHI clone 13 cells, as well as other cell lines, tested in their respective media, were resistant. The lower sensitivity of the cell lines to hp-DHA in MEM-medium was apparently due to a more rapid reduction of hp-DHA to the corre-sponding hydroxy-DHA in MEM-medium. Addition of glutathione (GSH) to the culture medium abolished the effects of hp-DHA, but not the effects of DHA, while depletion of intracellular GSH levels by L-buthionine-S,R-sulfoximine strongly enhanced the cytotoxic effect of hp-DHA, but not the cytotoxic effect of DHA. α-Tocopherol protected A-427 cells against the toxic effect of DHA and abolished the induced lipid peroxidation, while it did not protect against the toxic effects of hp-DHA in A-427 or WEHI clone 13 cells. Ascorbic acid reduced the cytotoxic effect of DHA, but potentiated the toxic effect of hp-DHA while selenite essentially abolished the toxicity of both DHA and hp-DHA. These results indicate that sensitivity of tumor cell lines to PUFA and their oxidation products depends on their antioxidant defense mechanisms, as well as culture conditions, and establishes hp-DHA as a major, but probably not the sole, metabolite responsible for cytotoxicity of DHA.