The Salmonella Enteritidis TolC outer membrane channel is essential for egg white survival.

Salmonella Enteritidis has developed the potential to contaminate eggs by surviving in the antimicrobial environment of the hen's egg white. This has led to a worldwide pandemic of foodborne salmonellosis infections in humans due to the consumption of contaminated eggs and egg-derived products. The molecular mechanisms of Salmonella Enteritidis egg white survival are not fully clear. Using in vivo expression technology and promoter-reporter fusions we showed that the promoter of the tolC gene, encoding the TolC outer membrane channel that is used by multidrug efflux pumps to export harmful molecules and to secrete bacterial products, is activated by egg white at the chicken body temperature. Using a Salmonella Enteritidis tolC deletion mutant we showed that TolC has an important role in egg white survival. Chromatographic separation techniques and subsequent testing of antimicrobial activities of separated egg white fractions led to the identification of ovotransferrin as the egg white antimicrobial factor which is capable of inhibiting growth of a tolC deletion strain but not the wild type strain. We provide evidence that TolC protects Salmonella Enteritidis against ovotransferrin-mediated growth inhibition in egg white.

Evaluation of post-procedure changes in aneurysmal lumen following detachable coil-placement using multi-planar reconstruction of high-field (3.0T) magnetic resonance angiography.

BACKGROUND: Placement of detachable coil(s) for intracranial aneurysms has become one of the standard methods of management. Although detailed analysis of post-procedure changes in aneurysmal lumen is essential, technical difficulties often limit such evaluation. Development of higher magnetic field systems is steadily widening clinical usage of magnetic resonance imaging (MRI) primarily due to its significantly higher signal to noise ratio. OBJECTIVE: In this study, we evaluated a multi-planar reconstruction (MPR) technique of magnetic resonance angiography (MRA) on a 3.0T system in an attempt to develop a routine method of post-procedure evaluation following detachable coil placement. METHODS: Eleven patients with an intracranial aneurysm following placement of a Guglielmi detachable coil (GDC) participated in the study. Time of flight (TOF) magnetic resonance angiography (MRA) was obtained immediately after, and up to two years after coil embolisation utilising a GE 3.0T system. Data was analysed using standard maximum intensity projection (MIP) as well as the MPR technique and the results were compared to conventional catheter angiography. RESULTS: The study demonstrated that, compared to MIP, MPR can provide further information of alteration in aneurysm lumen, especially in analysis of: 1) jet of blood flow, 2) thrombus formation, 3) neck remnant or re-filling of blood, 4) location and shape of coils including compaction, and 5) coil protrusion into the parent artery. CONCLUSIONS: Combined MPR/MIP analysis of high-field MRA appears to be a powerful non-invasive method for evaluating GDC-treatment that can potentially replace conventional catheter angiography in many clinical situations.

Rapid transcriptional activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy. Evidence for brain natriuretic peptide as an "emergency" cardiac hormone against ventricular overload.

We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. To assess the pathophysiological role of BNP in ventricular overload, we have examined the gene expression of BNP, In comparison with that of ANP, in a model of cardiac hypertrophy using cultured neonatal rat ventricular cardiocytes. During cardiocyte hypertrophy evoked by endothelin-1, Phenylephrine, or PMA, the steady state level of BNP mRNA increased as rapidly as the "immediate-early" induction of the c-fos gene expression, and reached a maximal level within 1 h. Actinomycin D, a transcriptional inhibitor, completely diminished the response, while the translational blocked with cycloheximide did not inhibit it. In contrast, ANP mRNA began to increase 3 h after the stimulation, and accumulated during cardiocyte hypertrophy. The BNP secretion from ventricular cardiocytes was also stimulated, more rapidly than the ANP secretion. Furthermore, the turnover of BNP mRNA was significantly faster than that of ANP mRNA, being consistent with the existence of AUUUA motif in the 3'-untranslated region of BNP mRNA. These results demonstrate that the gene expression of BNP is distinctly regulated from that of ANP at transcriptional and posttranscriptional levels, and indicate that the characteristics of the BNP gene expression are suitable for its possible role as an " emergency" cardiac hormone against ventricular overload.

3D digital subtraction angiography of intracranial aneurysms: comparison of flat panel detector with conventional image intensifier TV system using a vascular phantom.

BACKGROUND AND PURPOSE: Compared with the image intensifier (I.I.)-TV system, the flat panel detector (FPD) system of direct conversion type has several theoretic advantages, such as higher spatial resolution, wide dynamic range, and no image distortion. The purpose of this study was to compare the image quality of 3D digital subtraction angiography (DSA) in the FPD and conventional I.I.-TV systems using a vascular phantom. MATERIALS AND METHODS: An anthropomorphic vascular phantom was designed to simulate the various intracranial aneurysms with aneurysmal bleb. The tubes of this vascular phantom were filled with 2 concentrations of contrast material (300 and 150 mg I/mL), and we obtained 3D DSA using the FPD and I.I.-TV systems. First, 2 blinded radiologists compared the volume-rendering images for 3D DSA on the FPD and I.I.-TV systems, looking for pseudostenosis artifacts. Then, 2 other radiologists independently evaluated both systems for the depiction of the simulated aneurysm and aneurysmal bleb using a 5-point scale. RESULTS: For the degree of the pseudostenosis artifacts at the M1 segment of the middle cerebral artery at 300 mg I/mL, 3D DSA with FPD system showed mild stenoses, whereas severe stenoses were observed at 3D DSA with I.I.-TV system. At both concentrations, the FPD system was significantly superior to I.I.-TV system regarding the depiction of aneurysm and aneurysmal bleb. CONCLUSION: Compared with the I.I.-TV system, the FPD system could create high-resolution 3D DSA combined with a reduction of the pseudostenosis artifacts.

Reduction of radiation dose for cerebral angiography using flat panel detector of direct conversion type: a vascular phantom study.

BACKGROUND AND PURPOSE: Compared with image intensifier television (I.I.-TV) system, an angiography system using the flat panel detector (FPD) of direct conversion type has a high spatial resolution, which may improve image quality, reduce patient exposure, or both. Our purpose was to evaluate the detection of simulated aneurysmal blebs under dose reduction with the FPD system in comparison with the I.I.-TV system. MATERIALS AND METHODS: A vascular phantom was designed to simulate various intracranial aneurysms with and without blebs, and this phantom was filled with 3 different concentrations of contrast material (300, 150, and 100 mg I/mL). 2D digital subtraction angiography (DSA) at low-dose mode of FPD system was compared with 2D DSA at a standard-dose mode of FPD system and a conventional mode of I.I.-TV system. Data analysis was based on 171 observations (57 aneurysms [20 with bleb and 37 without bleb] x 3 contrast material concentrations) by each of 7 radiologists, and the detection performances of blebs were compared using a receiver operating characteristic (ROC) analysis. RESULTS: The mean dose measurements with a phantom during 2D DSA were 0.36 mGy/frame with low-dose mode of FPD system, 0.72 mGy/frame with standard-dose mode of FPD system and 0.76 mGy/frame with I.I.-TV system. The mean Az at 100 mg I/mL was significantly higher for low-dose mode of FPD than for conventional-dose mode of I.I.-TV mean Az, 0.85 versus 0.56; P < .01), though differences were not significant with 150 and 300 mg I/mL between both systems. CONCLUSION: The FPD system allows a considerable dose reduction during 2D DSA without loss of the image quality.

A cone-beam volume CT using a 3D angiography system with a flat panel detector of direct conversion type: usefulness for superselective intra-arterial chemotherapy for head and neck tumors.

BACKGROUND AND PURPOSE: The development of flat panel detectors (FPDs) has made cone-beam CT feasible for practical use in a clinical setting. Our purpose was to assess the usefulness of cone-beam CT using the FPD in conjunction with conventional digital subtraction angiography (DSA) for performing superselective intra-arterial chemotherapy for head and neck tumors. MATERIALS AND METHODS: Twenty-three consecutive patients (43 feeding arteries) were prospectively examined. All of the patients underwent intra-arterial rotational angiography using an FPD system, and the cone-beam CT was reconstructed from the volume dataset. Two radiologists evaluated the quality of the cone-beam CT and then evaluated whether the additional information provided by the cone-beam CT was useful for the interventional procedures. RESULTS: In 41 (95%) of 43 arteries, the extent of contrast material perfusion was sufficiently visualized on cone-beam CT. In 20 (47%) of 43 arteries, the DSA plus cone-beam CT was superior to the DSA alone regarding the precise understanding of vascular territory of each artery. This information was helpful for predicting the drug delivery for superselective intra-arterial chemotherapy, especially in deeply invasive tumors with multiple feeding arteries. CONCLUSION: In superselective intra-arterial chemotherapy for head and neck tumors, cone-beam CT with FPD provides useful additional information, which allows interventional radiologists to determine the feeders, as well as the dose of antitumor agent for each feeder.

Application of the Cre recombinase/loxP system further enhances antitumor effects in cell type-specific gene therapy against carcinoembryonic antigen-producing cancer.

A considerable number of studies of cancer have shown that the cell type-specific promoter is an effective tool for selective expression of foreign genes in tumor cells. However, few reports have demonstrated significant in vivo antitumor effects using this strategy thus far, possibly because the low activity of such a promoter results in insufficient expression of genes in cancer cells as well as in insignificant antitumor effects, even when the cells are infected by highly efficient gene transfer methods. To overcome this problem, we used the Cre/loxP system for the cell type-specific gene therapy against carcinoembryonic antigen (CEA)-producing cancer. We constructed a pair of recombinant Ads. One expresses the Cre recombinase (Cre) gene under the control of the CEA promoter (Ad.CEA-Cre). The other contains the herpes simplex virus thymidine kinase (HSV-TK) gene separated from the strong CAG promoter by insertion of the neomycin resistance (neo) gene (Ad.lox-TK). The HSV-TK gene of the latter Ad is designed to be activated through excisional deletion of the neo gene by Cre enzyme released from the former one only when CEA-producing cells are infected simultaneously with these Ads. Coinfection by these Ads rendered a human CEA-producing cancer cell line 8.4-fold more sensitive to ganciclovir (GCV) compared with infection by Ad.CEA-TK alone, the HSV-TK gene of which is directly regulated by the CEA promoter. On the other hand, coinfection with these Ads did not significantly change the GCV sensitivity of non-CEA-producing cells. Intratumoral injection of Ad.CEA-Cre combined with Ad.lox-TK followed by GCV treatment almost completely eradicated CEA-producing tumors established in the subcutis of athymic mice, whereas intratumoral injection of Ad.CEA-TK with GCV administration at most retarded the growth of inoculated tumors. These results suggest distinct advantages of the Cre/loxP system applied in the conventional cell type-specific gene therapy against cancer.

Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells.

Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.

Light source position and reflectance estimation from a single view without the distant illumination assumption.

Several techniques have been developed for recovering reflectance properties of real surfaces under unknown illumination. However, in most cases, those techniques assume that the light sources are located at inifinity, which cannot be applied safely to, for example, reflectance modeling of indoor environments. In this paper, we propose two types of methods to estimate the surface reflectance property of an object, as well as the position of a light source from a single view without the distant illumination assumption, thus relaxing the conditions in the previous methods. Given a real image and a 3D geometric model of an object with specular reflection as inputs, the first method estimates the light source position by fitting to the Lambertian diffuse component, while separating the specular and diffuse components by using an iterative relaxation scheme. Our second method extends that first method by using as input a specular component image, which is acquired by analyzing multiple polarization images taken from a single view, thus removing its constraints on the diffuse reflectance property. This method simultaneously recovers the reflectance properties and the light source positions by optimizing the linearity of a log-transformed Torrance-Sparrow model. By estimating the object's reflectance property and the light source position, we can freely generate synthetic images of the target object under arbitrary lighting conditions with not only source direction modification but also source-surface distance modification. Experimental results show the accuracy of our estimation framework.

Adaptively merging large-scale range data with reflectance properties.

In this paper, we tackle the problem of geometric and photometric modeling of large intricately shaped objects. Typical target objects we consider are cultural heritage objects. When constructing models of such objects, we are faced with several important issues that have not been addressed in the past-issues that mainly arise due to the large amount of data that has to be handled. We propose two novel approaches to efficiently handle such large amounts of data: A highly adaptive algorithm for merging range images and an adaptive nearest-neighbor search to be used with the algorithm. We construct an integrated mesh model of the target object in adaptive resolution, taking into account the geometric and/or photometric attributes associated with the range images. We use surface curvature for the geometric attributes and (laser) reflectance values for the photometric attributes. This adaptive merging framework leads to a significant reduction in the necessary amount of computational resources. Furthermore, the resulting adaptive mesh models can be of great use for applications such as texture mapping, as we will briefly demonstrate. Additionally, we propose an additional test for the k-d tree nearest-neighbor search algorithm. Our approach successfully omits back-tracking, which is controlled adaptively depending on the distance to the nearest neighbor. Since the main consumption of computational cost lies in the nearest-neighbor search, the proposed algorithm leads to a significant speed-up of the whole merging process. In this paper, we present the theories and algorithms of our approaches with pseudo code and apply them to several real objects, including large-scale cultural assets.

Hypocalcemic effect of physiological concentrations of corticosterone in adrenalectomized-parathyroidectomized rats.

We have confirmed the serum calcium-raising effect of adrenalectomy in young male rats 5-6 h after parathyroidectomy that was first observed by others many years ago. (The phenomenon has also been reported in cats, dogs, and mice.) In addition, we have shown that adrenalectomy raises the serum ionized calcium as well as total calcium and that the effect occurs in young female as well as in young male rats. Furthermore, we have found that the serum calcium-raising effect of adrenalectomy occurs if the adrenalectomy is performed several days before parathyroidectomy or 6 h after parathyroidectomy, as well as at the same time as the parathyroidectomy. When the rats were adrenalectomized 7-9 days before parathyroidectomy and given small daily life-maintaining doses of corticosterone or cortisone acetate, this glucocorticoid treatment did not reverse the adrenalectomy effect. This led us to think at first that the effect of adrenalectomy must be due to the loss of an unknown adrenal factor rather than to loss of glucocorticoid. Additional experiments, however, in which corticosterone or hydrocortisone was administered by continuous release pellets, demonstrated conclusively that a small continuous supply of corticosterone (within the physiological range as determined by immunoassay of plasma) was sufficient to reverse the adrenalectomy effect. The results with hydrocortisone were similar at even lower doses than of corticosterone. Somewhat higher doses of corticosterone or hydrocortisone reduced the serum calcium even below the parathyroidectomy level. In a preliminary investigation of the specificity of the glucocorticoid effect we found that aldosterone, dehydroepiandrosterone, or estradiol had no effect on serum calcium under similar conditions. We conclude that the fall in serum calcium after parathyroidectomy in rats is due in part to the hypocalcemic effect of endogenous corticosterone. Thus, the loss of corticosterone after adrenalectomy explains the serum calcium-raising effect of adrenalectomy in parathyroidectomized rats. These results also suggest that glucocorticoids at physiological levels have a significant effect on calcium metabolism in general.

Rapid reduction in [3H]prazosin binding to gerbil forebrain membranes during bilateral common carotid artery occlusion.

Transient cerebral ischemia results in selective neuronal cell death. The mechanisms underlying this selective vulnerability to ischemia are only beginning to be elucidated. We studied the effect of ischemia on alpha 1-adrenergic receptor binding by measuring [3H]prazosin binding in gerbil forebrain membranes after 10 min of bilateral carotid occlusion. Binding was reduced from 62 +/- 3 to 33 +/- 4 fmol/mg protein. Binding in the same membranes to beta 2-adrenergic receptors were also decreased, but not to the extent of that to alpha 1-adrenergic receptors. Binding to muscarinic cholinergic [( 3H]quinuclydil benzilate) and beta 1-adrenergic receptors were only slightly depressed. Surprisingly, the protein content was significantly increased in the membrane fraction studied from ischemic forebrain (68 +/- 4 mg/g wet weight) compared with sham operated controls (57 +/- 4). The dramatic decrease in alpha 1-adrenergic receptor binding during ischemia is consistent with receptor binding studies of membranes pretreated with phospholipase A2 in vitro. It is not clear what effect this change in alpha 1-adrenergic receptor binding has on subsequent selective neuronal death. The recent demonstration that catecholamines and locus ceruleus neurons influence the loss of CA1 neurons in the hippocampus suggests that it may play an important modulatory role.

Hydroxyl radical generation in skeletal muscle atrophied by immobilization.

Male Wistar rats (15 weeks old), the ankle joints of one hindlimb of which were immobilized in the extended position for 12 days, were injected with salicylate as a trapping agent for hydroxyl radicals before sacrifice. Atrophied and contralateral soleus, typical slow red muscles were collected and their levels of 2,3-dihydroxybenzoic acid (2,3-DHB), one of the main reaction products formed by the attack of hydroxyl radical on salicylate, were determined using high-performance liquid chromatography with an electrochemical detector. There was a significant increase in 2,3-DHB in the atrophied muscle. This result strongly suggests the enhanced generation in vivo of hydroxyl radicals in atrophied muscle.

Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds.

DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. DNA strand breaks in cultured cells exposed to nickel compounds were measured by using a pulsed field gel electrophoresis technique. Among nickel compounds (Ni(3)S(2), NiO (black), NiO (green), and NiSO(4)), only Ni(3)S(2), which is highly carcinogenic, induced lesions of both double- and single-stranded DNA in cultured human cells (Raji and HeLa cells). Treatment of cultured HeLa cells with Ni(3)S(2) (10 microg/ml) induced a 1.5-fold increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) compared with control, whereas NiO (black), NiO (green), and NiSO(4) did not enhance the generation of 8-OH-dG. Intratracheal instillation of Ni(3)S(2), NiO(black), and NiO(green) to Wistar rats increased 8-OH-dG in the lungs significantly. NiSO(4) induced a smaller but significant increase in 8-OH-dG. Histological studies showed that all the nickel compounds used induced inflammation in lungs of the rats. Nitric oxide (NO) generation in phagocytic cells induced by Ni(3)S(2), NiO(black), and NiO(green) was examined using macrophage cell line RAW 264.7 cells. NO generation in RAW 264.7 cells stimulated with lipopolysaccharide was enhanced by all nickel particles. Two mechanisms for nickel-induced oxidative DNA damage have been proposed as follows: all the nickel compounds used induced indirect damage through inflammation, and Ni(3)S(2) also showed direct oxidative DNA damage through H(2)O(2) formation. This double action may explain relatively high carcinogenic risk of Ni(3)S(2).

Effect of chronic alcohol administration on transketolase in the brain and the liver of rats.

To estimate the nutritional and the pathological states in thiamin-deficiency-related diseases, especially Wernicke-Korsakoff syndrome, we studied the relationship among transketolase activity, transketolase concentration, and thiamin phosphate esters in rats chronically fed alcohol. In the brain of alcohol-fed rats, the enzyme activity and concentration decreased although there was no positive correlation between the two. On the contrary, transketolase activity in the liver correlated positively with concentration, and both transketolase activity and concentration were decreased in the thiamin-deficient groups. These findings suggest that transketolase in the brain may be different from that in the liver and that the alteration of the enzyme activity in the brain may be based on the conformational change of the protein molecule caused by chronic alcohol administration.

Synthesis and antiulcer activity of N-substituted N'-[3-[3-(piperidinomethyl)phenoxy]propyl]ureas: histamine H2-receptor antagonists with a potent mucosal protective activity.

As an aim toward developing new antiulcer agents, new N-substituted N'-[3-[3-(piperidinomethyl)phenoxy]propyl]ureas were synthesized and evaluated for histamine H2-receptor antagonistic, gastric antisecretory, and gastric mucosal protective activities. A QSAR study showed that the most favorable N-substituents were electron-donating straight-chain alkyl groups of short length such as ethyl group from the viewpoint of dual action, i.e., gastric antisecretory and mucosal protective actions. Among the ureas studied, compounds 4, 5, and 8-10 were selected as candidates for further study.

DSP4 treatment worsens hippocampal pyramidal cell damage after transient ischemia.

Recent studies in the rat have suggested that hippocampal norepinephrine can regulate the amount of damage seen after transient forebrain ischemia. We used the gerbil to study the role of norepinephrine in ischemic damage. Using tyrosine hydroxylase immunocytochemistry and chemical measurements of norepinephrine, we determined that the gerbil hippocampus has a similar but topographically different norepinephrine innervation than the rat. Brains from gerbils treated with 100 mg/kg of N-(2-chloroethyl)-N-methyl-2-bromobenzylamine (DSP4) had 60% less norepinephrine than saline-treated controls, similar to the effect of the drug in rats. We administered DSP4 to gerbils two weeks before exposing them to 5 min of bilateral carotid artery occlusion. Animals treated with DSP4 and subjected to ischemia had worse pyramidal cell loss in the CA3 and CA4 regions than saline-treated ischemic controls. CA1 pyramidal cell loss (about 90%) was severe in both the saline- and DSP4-treated animals. These data provide further evidence that norepinephrine can regulate the neuronal death in the hippocampal formation after transient forebrain ischemia. Furthermore, this is the first demonstration of that regulation in the gerbil and suggests that noradrenergic input to the hippocampus may be important in ischemia in other species besides the rat.

Antioxidant action of the antihypertensive drug, carvedilol, against lipid peroxidation.

The action of carvedilol, a vasodilating, beta-adrenoceptor blocking agent, against lipid peroxidation has been the subject of many studies, but the results reported thus far are contradictory. In an attempt to define the antioxidant mechanism of carvedilol against lipid peroxidation, the dynamics of the action of carvedilol were studied in several oxidation systems. We investigated the reactivity of carvedilol toward radicals and its inhibitory effect on lipid peroxidation induced by several kinds of initiating species such as azo compounds and metal ions in solution, micelles, membranes, and low-density lipoprotein. Carvedilol exerted poor reactivity toward phenoxyl, alkoxyl, and peroxyl radicals in acetonitrile solution nor did it show an appreciable antioxidant effect against either the peroxyl radical-induced oxidation of methyl linoleate in acetonitrile or against phosphatidylcholine liposomal membranes in aqueous suspension. Carvedilol completely inhibited the ferric ion-induced oxidation of methyl linoleate micelles by sequestering ferric ions, but not by reducing hydroperoxide. It was shown that carvedilol enhanced the oxidation of micelles induced by either methemoglobin or peroxyl radical. Carvedilol, which was added exogenously, did not suppress the oxidation of isolated low-density lipoprotein induced by peroxyl radical or cupric ion. These results show that carvedilol does not act as a radical-scavenging antioxidant, but that it does act most efficiently as an antioxidant against ferric ion-induced oxidation by sequestering ferric ion.

Oxidative DNA damage and apoptosis induced by metabolites of butylated hydroxytoluene.

DNA damage by metabolites of a food additive, butylated hydroxytoluene (BHT), was investigated as a potential mechanism of carcinogenicity. The mechanism of DNA damage by 2,6-di-tert-butyl-p-benzoquinone (BHT-quinone), 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHT-OOH), and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) in the presence of metal ions was investigated by using 32P-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. BHT-OOH caused DNA damage in the presence of Cu(II), whereas BHT-quinone and BHT-CHO did not. However, BHT-quinone did induce DNA damage in the presence of NADH and Cu(II). Bathocuproine inhibited Cu(II)-mediated DNA damage, indicating the participation of Cu(I) in the process. Catalase also inhibited DNA damage induced by BHT-quinone, but not that induced by BHT-OOH. The DNA cleavage pattern observed with BHT-quinone plus NADH was different from that seen with BHT-OOH. With BHT-quinone plus NADH, piperidine-labile sites could be generated at nucleotides other than adenine residue. BHT-OOH caused cleavage specifically at guanine residues. Pulsed field gel electrophoresis showed that BHT-OOH and BHT-quinone induced DNA strand breaks in cultured cells, whereas BHT-CHO did not. Both BHT-quinone and BHT-OOH induced internucleosomal DNA fragmentation, which is the characteristic of apoptosis. Furthermore, flow cytometry analysis revealed an increase of peroxides in cultured cells treated with BHT-OOH or BHT-quinone. These results suggest that BHT-OOH participates in oxidative DNA damage directly, whereas BHT-quinone causes DNA damage through H2O2 generation, which leads to internucleosomal DNA fragmentation.