In vivo EPR detection and imaging of endogenous nitric oxide in lipopolysaccharide-treated mice.

Nitric oxide (NO), a simple diatomic free radical, is known to play a critical physiological role in diverse organisms. An iron complex, with N-(dithiocarboxy)sarcosine (Fe-DTCS), has a high affinity for endogenous NO and can trap, stabilize, and accumulate it. The stable NO adduct thus formed is detectable at room temperature with electron paramagnetic resonance (EPR) spectrometry. We report in vivo EPR imaging of endogenous NO, trapped by an Fe-DTCS complex, in the abdomen of a live mouse. To our knowledge, this is the first report on EPR imaging of endogenous free radicals produced in vivo. This EPR imaging method will be useful for the noninvasive investigation of the spatial distribution of NO in pathologic organs or tissues.

Innervation and functional changes in mesenteric perivascular calcitonin gene-related peptide- and neuropeptide Y-containing nerves following topical phenol treatment.

We have previously shown that age-related reduction of innervation and function in mesenteric perivascular calcitonin gene-related peptide-containing vasodilator nerves takes place in spontaneously hypertensive rats. The present study was performed to investigate innervation and functional changes in perivascular calcitonin gene-related peptide- and adrenergic neuropeptide Y-containing nerves after topical treatment with phenol, which damages nerve fibers, around the rat superior mesenteric artery. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) or saline (sham rats) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the 3rd branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 3 through day 14. The innervation levels of calcitonin gene-related peptide-like immunoreactivity containing fibers and neuropeptide Y-like immunoreactivity containing fibers were markedly reduced on day 3 to day 14 and on day 5 to day 14 after the treatment, compared with those in sham-operated rats, respectively. In perfused mesenteric vascular beds isolated from phenol-treated rats, adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide nerve-mediated vasodilation in response to periarterial nerve stimulation (2-12 Hz) were significantly decreased on day 3 and day 7. Neurogenic release of norepinephrine in phenol-treated rats on day 7 was significantly smaller that that in sham-operated rats. Nerve growth factor content in the mesenteric arteries of phenol-treated rats was significantly lower than that in sham-operated rats. Administration of nerve growth factor using osmotic mini-pumps for 7 days after the phenol treatment resulted in greater density of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity fibers than in phenol-treated rats and restored decreased vascular responses to periarterial nerve stimulation. These results suggest that topical phenol-treatment of the mesenteric artery effectively induces functional denervation of perivascular nerves, which can be prevented or reversed by nerve growth factor treatment.

Possible link between glycolysis and apoptosis induced by sodium fluoride.

Fluoride has been used to prevent caries in the dentition, but the possible underlying mechanisms of cytotoxicity induction by this compound are still unclear. Since fluoride is known as an inhibitor of glycolytic enzymes, we investigated the possible connection between NaF-induced apoptosis and glycolysis in human promyelocytic leukemia HL-60 cells. NaF-induced apoptotic cell death is characterized by caspase activation, internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, and production of apoptotic bodies. Higher activation of caspases-3 and -9, as compared with that of caspase-8, suggested the involvement of an extrinsic pathway. Utilization of glucose was nearly halted by NaF, whereas that of glutamine was rather enhanced. NaF enhanced the expression of Bad protein, but not that of Bcl-2 and Bax proteins, and reduced HIF-1alpha mRNA expression. Analysis of these data suggests a possible link between glycolysis and apoptosis.

Lipo-PGE1, prostaglandin E1 incorporated in lipid microspheres, protects injury of the liver caused by warm ischemia reperfusion.

Effects of Lipo-PGE1, prostaglandin E1 incorporated in lipid microspheres on liver injury caused by ischemia reperfusion were investigated. Lipo-PGE1 (10 micrograms/kg or 3 micrograms/kg) or vehicle was gradually injected twice via portal vein 5 min prior to induction of ischemia and reperfusion. Rats died within 2 d after liver ischemia of 90 min from the group receiving injection of vehicle alone. Lipo-PGE1 had its most profound effect on the survival of animals subjected to liver ischemia followed by reperfusion when given in two doses, one prior to ischemia, and another prior to reperfusion. Lipo-PGE1 markedly suppressed both the increases in plasma PCOOH (phosphatidyl-choline hydroperoxide) levels and the leakage of GOT, GPT, and LDH from the liver during the ischemia reperfusion. These findings suggest that Lipo-PGE1 may have therapeutic applications in treatment of hepatic injury.

Developmental changes in and hormonal modulation of epidermal growth factor concentration in the rat submandibular gland.

Developmental changes in the hormonal effects on the concentration of epidermal growth factor (EGF) in the rat submandibular gland were investigated. The level of EGF in the gland gradually increased with age from 4 up to 8 weeks of age; thereafter it increased markedly, reaching a plateau level at 12 weeks of age in both male and female rats. A significant sex difference in EGF levels was observed between 8 and 14 weeks, the level in the males being approximately twice as high as that in the females at 12 and 14 weeks of age. Castration of male rats decreased EGF to about the same level as that of control females. Treatment of castrated rats with testosterone propionate (TP) restored EGF to the levels in control male rats. Ovariectomy and/or administration of oestradiol-17 beta to ovariectomized rats had no apparent effect on EGF concentration. These findings indicate that the sex difference in EGF concentration can be attributed to the level of endogenous androgens. In addition, hypophysectomy of male rats caused a remarkable decrease in submandibular gland EGF to about 7% of the normal level. This reduction was significantly, although not completely, restored by the administration of TP, triiodothyronine (T3) or GH. Moreover, giving TP with T3 or with GH or both together had additive effects on the increase in EGF levels in hypophysectomized rats. These results provide evidence that EGF in the submandibular gland is regulated multihormonally by at least TP, T3 and GH.

Chemiluminescence-HPLC assay of phosphatidylcholine hydroperoxide generated by ischemia-reperfusion in the liver of rats.

To determine cellular damage due to "oxidative stress", we developed a sensitive and specific quantitative assay for phosphatidylcholine hydroperoxide (PCOOH) by coupling HPLC with detection of chemiluminescence (CL). The qualitative and quantitative detection limits of PCOOH by this assay were 0.5 and 2 pmol (based on active oxygen from hydroperoxide). Using this CL-HPLC method, we determined PCOOH levels caused by ischemia-reperfusion in rat livers. The PCOOH levels in livers of control, sham-operated and operated rats with only ischemic treatment were approximately 2 nmol/g wet liver weight. The PCOOH level and several serum parameters of liver injury increased with an increase in the duration of ischemia, and also increased in proportion to the duration of reperfusion. The determination of PCOOH in liver caused by ischemia-reperfusion could be a useful method for investigating liver damage induced by free radicals.

Effects of anti-free radical interventions on phosphatidylcholine hydroperoxide in plasma after ischemia-reperfusion in the liver of rats.

The present study set out to investigate whether plasma phosphatidylcholine hydroperoxide (PCOOH) levels could accurately reflect lipid peroxidation linking to liver damage due to ischemia--reperfusion. PCOOH is a primary peroxidative product of phosphatidylcholine (PC), which is the most important functional lipid in the hepatocellular membrane, and may mediate oxidative stress. We quantified PCOOH and PC in the plasma and liver of rats subjected to hepatic ischemia-reperfusion by chemiluminescence detecting HPLC (CL-HPLC) method. Plasma PCOOH levels showed no significant rise in either the ischemia only group or in the sham-operation group, compared to controls (0.7 nmol/mL plasma). At 60 min subsequent to reperfusion, the PCOOH levels in plasma and liver, as well as the levels of several serum markers of liver injury [lactic dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT)] increased in proportion to the duration of ischemia (up to 60 min). During periods of reperfusion following 30 min of ischemia, plasma PCOOH increased biphasically (2 nmol/mL; 12-24 hr duration of reperfusion), and generally ran parallel to that in the liver after more than 60 min of reperfusion. Dose-dependent protective effects against warm ischemia (30 min)-reperfusion (12 hr) injury were clearly demonstrated in the groups treated with allopurinol, diclofenac Na, ascorbic acid (V.C), alpha-tocopherol and coenzyme Q10, but not in those treated with r-h-superoxide dismutase or betamethasone. The rises in plasma PCOOH and serum GOT, GPT and LDH of the ischemia-reperfused rats were ameliorated most in the group pretreated with diclofenac Na, and next most in the group pretreated with V.C. These results indicate that the plasma PCOOH levels are a useful index both for liver cell damage induced by oxygen free radicals generated during ischemia-reperfusion, and to investigate the efficacy of drugs against oxidative stress.

Head and neck lesions: determination of an optimal MT technique for prediction of malignancies.

RATIONALE AND OBJECTIVES: To determine an optimal magnetization transfer (MT) technique for diagnosis of malignancies in the head and neck. METHODS: Lesion magnetization transfer ratios (MTRs) and lesion-to-muscle MTRs were prospectively measured in 52 head and neck lesions of 52 patients at frequency offsets of 0.3, 0.5, and 1 kHz from water resonance. The diagnostic capability for each MT pulse was calculated by using receiver operating characteristic (ROC) curves, and an optimal MT technique was determined for diagnosis of malignancies. RESULTS: The lesion MTRs and the lesion-to-muscle MTRs in malignant tumors were statistically significantly greater than those in benign lesions at both 0.5- and 1-kHz MT pulses, but no significant differences were noted between them at the 0.3-kHz MT pulse. Diagnosis with the lesion-to-muscle MTRs was better than that with the lesion MTRs at each MT pulse. Among all MTRs, lesion-to-muscle MTRs at the 1-kHz MT pulse showed the highest diagnostic capability for malignancies (area under the ROC curve = 0.82+/-0.06). With a lesion-to-muscle MTR at a 1-kHz MT pulse of 0.61 or greater, the highest accuracy of 85% was attained with 90% sensitivity and 77% specificity. CONCLUSIONS: Lesion-to-muscle MTRs at a 1-kHz MT pulse were optimal for diagnosis of malignancies in the head and neck.

Glutathionyl hemoglobin in uremic patients undergoing hemodialysis and continuous ambulatory peritoneal dialysis.

BACKGROUND: To assess the redox state in hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients, we focused on the formation of glutathionyl hemoglobin (Hb) because the ratio of oxidized glutathione disulfide (GSSG) to reduced glutathione (GSH) is increased in uremia, and GSSG is a source of glutathionyl Hb. METHODS: Glutathionyl Hb levels were measured in 30 HD patients, 10 CAPD patients, and 20 healthy subjects by using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). RESULTS: Hbbeta showed a peak at 15,868 D in a deconvoluted ESI mass spectrum. Glutathionyl Hbbeta was detected at 16,173 D (15,868 + 305). The peak at 16,173 D was identified as glutathionyl Hbbeta based on the following findings: (1) the peak disappeared by reducing the sample with dithiothreitol, and (2) the peak could be detected at a high level by incubating Hb in vitro with GSH in water at 37 degrees C for seven days. Glutathionyl Hb levels expressed as the peak height ratios of glutathionyl Hbbeta to intact Hbbeta were significantly elevated in HD patients (8.0 +/- 3.6%, mean +/- SD, N = 30, P < 0.0001) and CAPD patients (5.9 +/- 2.7%, N = 10, P < 0.05) as compared with normal subjects (3.0 +/- 1.6%, N = 20). However, there were no significant differences in the glutathionyl Hb levels before (8.7 +/- 3.2%, N = 12) and after HD (8.7 +/- 2.8%, N = 12). CONCLUSION: Glutathionyl Hb levels were increased in HD and CAPD patients, probably because of enhanced oxidative stress. The measurement of glutathionyl Hb may be useful to assess oxidative stress in uremic patients.

Dialysis-related amyloidosis of the heart in long-term hemodialysis patients.

BACKGROUND: Dialysis-related amyloidosis (DRA) predominantly occurs in the osteoarticular structures, but it also systemically appears in the extra-articular tissues as well. However, the pathological characteristics of DRA in the hearts of hemodialysis (HD) patients have rarely been reported. METHODS: We studied the pathological characteristics of DRA in the hearts of 18 HD patients, including its relationship to calcification. Furthermore, we studied the immunohistochemical localization of advanced glycation end products (AGEs) using monoclonal anti-imidazolone and anti-Nepsilon-(carboxymethyl)lysine (CML) antibodies. RESULTS: beta2-microglobulin (beta2m) amyloid deposits were detected in the hearts of seven patients who had undergone HD for more than 10 years. beta2m amyloid deposits in the left atrium were localized in the endocardium, the myocardium, and the walls of small blood vessels, whereas in the left ventricle, they were localized only in the walls of small blood vessels. The extent and prevalence of DRA in the heart were severe in the patients on HD for more than 15 years. Most calcification areas near mitral valve were dotted with beta2m amyloid deposits, while diffuse fine calcification was localized within the beta2m amyloid tissues in some cases. Imidazolone and CML were localized not only in massive beta2m amyloid deposits, but also in cardiac myocytes. CONCLUSION: DRAs were localized extensively in the hearts of long-term HD patients. A strong affinity was observed between beta2m amyloid deposits and calcification.

Oral adsorbent ameliorates renal TGF-beta 1 expression in hypercholesterolemic rats.

BACKGROUND: A spontaneously hypercholesterolemic Imai rat has recently been reported as a model of focal glomerulosclerosis that causes nephrotic syndrome followed by renal failure. This study was designed to determine if an oral adsorbent, AST-120, ameliorates renal lesions and TGF-beta 1 expression in the rats. METHODS: AST-120 was given orally to the Imai rats for 32 weeks, and renal function and pathology were compared between the AST-120-administered and control Imai rats. RESULTS: AST-120-administered rats showed significantly lower level of blood urea nitrogen, serum creatinine, urinary protein, serum total-cholesterol, serum triglyceride, and serum and urinary indoxyl sulfate, and significantly higher levels of serum albumin and creatinine clearance than control rats. AST-120 reduced the glomerular sclerosis index, interstitial fibrosis area, and the extent of glomerular lipid deposition. Immunohistochemistry demonstrated that AST-120 reduced the expression of transforming growth factor (TGF)-beta 1 and tissue inhibitor of metalloproteinase (TIMP)-1 as well as interstitial infiltration of macrophages in the renal cortex of the Imai rats. CONCLUSIONS: AST-120 prevented the progression of nephrotic syndrome and renal failure in the Imai rats by ameliorating glomerular sclerosis and interstitial fibrosis, accompanied with reduced expression of TGF-beta 1 and TIMP-1, and reduced infiltration of macrophages in the kidneys.

Effect of endotoxin-induced reactive oxygen species on sperm motility.

OBJECTIVE: To define the mechanism of infection-induced damage of sperm. DESIGN: The effect of lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) on sperm motility and its modification by scavengers were investigated. SETTING: Research laboratory of a university hospital. PATIENT(S): Normozoospermic semen samples were obtained from 37 healthy volunteers. INTERVENTION(S): The sperms were incubated in the presence of LPS with or without scavengers. MAIN OUTCOME MEASURE(S): Sperm motility was evaluated by a sperm quality analyzer (SQAIIB). ROS formation in semen samples was measured by a Berthold luminometer (LB953). RESULT(S): Motility of spermatozoa was decreased in the LPS-treated samples compared with that in the control groups. ROS was significantly higher in the LPS-treated groups than in the control groups. The addition of ROS scavengers restored the motility index and suppressed ROS production in the LPS-treated semen samples. CONCLUSION(S): These data suggest that endotoxin-induced excessive production of ROS is responsible for the decrease in sperm motility and that antioxidant therapy may be a therapeutic option for infertile men with bacterial genital tract infection.

Papillary thyroid carcinoma: MR diagnosis of lymph node metastasis.

PURPOSE: The purpose of this study was to ascertain the usefulness of MR imaging in the diagnosis of nodal metastasis of papillary thyroid carcinoma and to establish the most indicative MR criteria of metastasis. METHODS: Pathologic records and MR images in 50 patients with papillary thyroid carcinoma were reviewed. Each neck was divided into four nodal levels, so that 200 nodal levels were assessed in all. The maximum of the minimum transverse diameters of the lymph nodes on each nodal level measured on MR images and the certainty of metastasis as determined by a head and neck radiologist on the basis of morphologic aspects were compared with the pathologic findings by using receiver operating characteristic curves. The presence or absence of cystic nodes on each nodal level was also evaluated. RESULTS: Metastasis was found on 87 (44%) of the nodal levels in 34 (68%) of the patients. A cystic node was identified on 33 (17%) of the nodal levels in 13 (26%) of the patients and was seen only on positive nodal levels. Morphologic diagnosis by the radiologist was better than that obtained by measurement. With the combined criteria of a cystic node or a node of 13 mm or more for the maximum of the minimum transverse diameters, specificity was 100% with an 82% accuracy and always indicated metastasis (100% positive predictive value). However, 41% of the metastatic nodes were missed with this criterion (59% sensitivity). CONCLUSION: MR imaging was useful for diagnosing metastatic nodes; a nodal diameter threshold of 13 mm or the presence of a cystic node strongly indicated metastasis from papillary thyroid carcinoma.

Primary thyroid lymphoma: diagnosis of immunoglobulin heavy chain gene rearrangement with polymerase chain reaction in ultrasound-guided fine-needle aspiration.

We present a case of primary thyroid lymphoma coexisting with Hashimoto's thyroiditis in a 75-year-old woman in whom B-cell lymphoma was substantiated based on the findings of immunophenotyping and polymerase chain reaction (PCR) gene rearrangement in specimens that had been obtained by ultrasound (US)-guided fine-needle aspiration biopsy (FNAB). The immunophenotyping technique showed A light chain restriction, and PCR-based assays showed a discrete narrow band, which was diagnostic for clonal B-cell proliferation. Analyses of PCR gene rearrangement in US-guided FNAB may be a useful ancillary technique to pathological findings for diagnosis of primary thyroid lymphoma, especially for differentiation between low-grade B-cell lymphomas and Hashimoto's thyroiditis.

Prognostic significance of magnetic resonance findings in advanced papillary thyroid cancer.

We assessed the prognostic importance of magnetic resonance (MR) findings in locally advanced papillary thyroid cancer. MR findings, clinical data, and pathologic (and surgical) data for 66 patients, including 51 women and 15 men with a mean age of 57 years, who had primary surgery for papillary thyroid cancers were correlated with prognosis. Mean follow-up was 27.5 months (range, 5-117 months). Recurrence was seen in 18 patients (27%). In univariate analyses, age of 60 years or more (p = 0.0066), male gender (p = 0.0373), six MR findings (tumor size of > or = 4 cm ([p = 0.0002], ill-defined margins ([p < 0.0001], tumor extension of the trachea [p = 0.0337], carotoid artery [p = 0.0028]), esophagus [p < 0.0001], and lymph nodes [p = 0.0005]), and three pathologic findings (tumor extension of soft tissues [p = 0.0288], carotid artery [p = 0.0013], and esophagus [p < 0.0001]) had a significant adverse effect on disease-free survival. In multivariate analyses, tumor size (p = 0.0169) and nodal metastasis (p = 0.0393) determined on MR imaging and pathologic esophageal invasion (p = 0.0016) were the only significant independent variables. Esophageal invasion was accurately diagnosed with MR imaging (94% accuracy). MR findings may contain prognostic importance of locally advanced papillary thyroid cancer.

Singlet oxygen generation from phosphatidylcholine hydroperoxide in the presence of copper.

This study pursued whether singlet oxygen ((1)O2) is generated from phosphatidylcholine hydroperoxide (PCOOH), the oxidized modification product of a major constituent of biomembranes and serum lipoproteins. The (1)O2 formation was detected, by utilizing the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TMPD) by (1)O2 to yield 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE), which generates electron spin resonance (ESR) signals. The TEMPONE signal was detected in human plasma with addition of PCOOH by ESR determination after introducing copper(II). The TEMPONE formation was proportional to the amounts of PCOOH added according to moles of active oxygen. The TEMPONE signal intensity was weakened significantly in the presence of beta-carotene and histidine in a concentration-dependent manner, but was not at all decreased by mannitol, Mn-superoxide dismutase and catalase. In addition, HPLC-chemiluminescence analysis demonstrated that incubation with the PCOOH/Cu(II) combination oxidized cholesterol, a relatively oxidation-resistant component, to the cholesterol hydroperoxide. These results reveal that (1)O2 is generated from PCOOH in contact with copper(II). In conclusion, this in-vitro study provides directly the (1)O2 formation in living organisms following the advancement of peroxidation of constitutive lipids.

Effect of antioxidants on radical intensity and cytotoxicity of hydroquinone.

Hydroquinone (HQ) dose-dependently reduced the viable cell number of oral tumor cell lines (HSC-2, HSG). HQ induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells, but not in HSC-2 nor HSG cells. Cytotoxic activity of HQ was slightly reduced by catalase, but was enhanced by superoxide dismutase, suggesting the possible involvement of hydrogen peroxide in HQ-induced cytotoxicity. This was supported by slight increase or decrease of cytotoxicity of HQ in the presence of Cu2+ and Fe3+, respectively. Lower concentrations of sodium ascorbate, ascorbic acid and ascorbic acid 6-palmitate reduced both the radical intensity and cytotoxic activity of HQ, more efficiently than ascorbic acid 2,6-dipalmitate, in contrast to the cytotoxic action of these ascorbates at higher (millimolar) concentrations. Popular antioxidants such as N-acetyl-L-cysteine and cysteine also reduced the radical intensity and cytotoxic activity of HQ. The present study suggests that cytotoxic activity of HQ is generated by radical-mediated oxidation mechanism.

Cytotoxic activity of steroidal saponins against human oral tumor cell lines.

Three steroidal saponins showed higher cytotoxicity against human oral squamous cell carcinoma cell lines (HSC-2), as compared with normal human gingival fibroblasts HGF. Tumor specificity of saponins exceeded that of tannins and flavonoids. Agarose gel electrophoresis showed that saponins failed to induce internucleosomal DNA fragmentation, but produced large DNA fragments in both HSC-2 cells and human promyelocytic leukemic HL-60 cells. In contrast to epigallocatechin gallate or gallic acid, cytotoxic activity of saponins was not significantly affected by metals (Co2+, Cu2+, Fe3+) nor by antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase). Furthermore, saponins did not produce radicals (detected by ESR spectroscopy) nor oxidation potential (measured by NO monitor). These data suggest that an oxidation-mediated mechanism is not involved in the cytotoxicity induced by steroidal saponins.

Cytotoxic activity of polyprenylalcohols and vitamin K2 derivatives.

Cytotoxic activity of 9 polyprenylalcohols and 6 vitamin K2 derivatives (MK-1 to MK-6) with various lengths of prenyl units was investigated. Among these compounds, geranylgeraniol with 4 prenyl units, and MK-2 with 2 prenyl units, showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), without induction of internucleosomal DNA fragmentation. Higher molecular weight compounds showed selective cytotoxicity against tumor cell lines than normal human gingival fibroblasts HGF. ESR spectroscopy showed that all polyprenylalcohols did not produce radical, nor scavenged O2- generated by hypoxanthine and xanthine oxidase reaction, and only slightly enhanced the radical intensity of sodium ascorbate. Vitamin K2 derivatives scavenged O2- more efficiently, but did not produce radical (except MK-3) and only slightly modified the ascorbate radical intensity. Cytotoxic activity of these compounds might be affected by the molecular weight, hydrophobicity, van der Waals area and stabilization of hydration of the molecule.

Cytotoxic activity of saponins from Camassia leichtlinii against human oral tumor cell lines.

Five steroidal saponins from Camassia leichtlinii showed higher cytotoxicity against human oral squamous cell carcinoma cells HSC-2, as compared to normal human gingival fibroblasts HGF. The tumor specificity of saponins varied considerably from sample to sample, but was generally higher than that of tannins, flavonoids and prenylated compounds such as geranylgeraniol and vitamin K2 (MK-2). Agarose gel electrophoresis showed that the saponins failed to induce internucleosomal DNA fragmentation, but produced large DNA fragments in HSC-2 cells, whereas two saponin samples (compounds 1 and 5) induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells. In contrast to epigallocatechin gallate or gallic acid, the cytotoxic activity of saponins was not significantly affected by metals (Co2+, Cu2+, Fe3+) or by antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase). Furthermore, the saponins did not produce radicals (detected by ESR spectroscopy) nor oxidation potential (measured by NO monitor). These data suggest that an oxidation-mediated mechanism is not involved in the cytotoxicity induced by the steroidal saponins.