Molecular signatures associated with cognitive deficits in schizophrenia: a study of biopsied olfactory neural epithelium.

Cognitive impairment is a key feature of schizophrenia (SZ) and determines functional outcome. Nonetheless, molecular signatures in neuronal tissues that associate with deficits are not well understood. We conducted nasal biopsy to obtain olfactory epithelium from patients with SZ and control subjects. The neural layers from the biopsied epithelium were enriched by laser-captured microdissection. We then performed an unbiased microarray expression study and implemented a systematic neuropsychological assessment on the same participants. The differentially regulated genes in SZ were further filtered based on correlation with neuropsychological traits. This strategy identified the SMAD 5 gene, and real-time quantitative PCR analysis also supports downregulation of the SMAD pathway in SZ. The SMAD pathway has been important in multiple tissues, including the role for neurodevelopment and bone formation. Here the involvement of the pathway in adult brain function is suggested. This exploratory study establishes a strategy to better identify neuronal molecular signatures that are potentially associated with mental illness and cognitive deficits. We propose that the SMAD pathway may be a novel target in addressing cognitive deficit of SZ in future studies.

Effect of modification of the carboxyl groups of the sialic acid binding lectin from bullfrog (Rana catesbeiana) oocyte on anti-tumor activity.

The sialic acid binding lectin from bullfrog Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity. In order to investigate the relationship between the net charge of cSBL and its anti-tumor effect, cSBL was modified with a water-soluble carbodiimide (EDC) in the presence of three kinds of nucleophiles, taurine, glycine methylester and ethylenediamine. cSBL having four carboxyl groups was partially modified (ca. 2 residues). The anti-tumor activity of modified cSBLs was in the order of ethylenediamine-modified cSBL > glycine methylester-modified cSBL > taurine modified cSBL > or = native cSBL. The results suggested that anti-tumor activity seems to increase with the increase in positive net charge, possibly enhancing the interaction of cSBL with sialoglycoprotein on the surface of tumor cells. The ribonuclease activity of ethylenediamine-modified cSBL decreased with the progress of the reaction, but the number of internalized molecules in the tumor cell increased. Thus, for antitumor activity, a higher incorporation of cSBL with reasonable RNase activity seems to be more important than total RNase activity.

Reversal of vinblastine resistance in human leukemic cells by haloperidol and dihydrohaloperidol.

Haloperidol, an antipsychotic, was investigated in cells overexpressing P-glycoprotein to detemine whether it was a clinically effective drug to reverse for reversing multidrug resistance (MDR) mediated by P-glycoprotein. A nontoxic concentration of haloperidol (1-30 microM) enhanced the cytotoxic effects of vinblastine (VBL) concentration-dependently in VBL-resistant human leukemia (K562/VBL) cells, but had no effect in the parent cells. Haloperidol also enhanced the cytotoxicities of epirubicin, doxorubicin and actinomycin D in the K562/VBL cells, but not those of idarubicin or cisplatin; this enhancement was less than that of the VBL toxicity in the VBL-resistant tumor line. Haloperidol increased the intracellular accumulation of VBL in the K562/VBL cells, and the binding of [3H]-azidopine to the cell-surface protein, P-glycoprotein, was inhibited by haloperidol in a concentration-dependent manner. Haloperidol was less potent than verapamil. Thus, haloperidol appeared to potentiate anticancer agents through the reversal of MDR by competitively inhibiting drug-binding to P-glycoprotein. In contrast, the main metabolite of haloperidol, dihydrohaloperidol, without antipsychotic activity, had less of an effect. Therefore, haloperidol might be useful in reversing drug-resistance.

Mechanism of resistance to oxidative stress in doxorubicin resistant cells.

Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.

Modification of tumor necrosis factor-induced acute toxicity D-galactosamine challenge by polymyxin B, an anti-endotoxin.

Polymyxin B (PMB), an antibiotic with anti-endotoxin activity, was used to examine the participation of endogenously produced endotoxin in the enhancement of recombinant human tumor necrosis factor (rhTNF)-induced toxicity in D-galactosamine (GalN)-sensitized mice. GalN-sensitized mice (700 mg/kg, intraperitoneally (i.p.)) injected together with rhTNF (1x10(4) U/mouse, intravenously (i.v.)) exhibited severe symptoms, with 100% mortality at 18 h. However, mice pretreated with PMB (20 mg/kg, i.p.) showed protection against the rhTNF-induced lethality following GalN sensitization. Little or no effects were observed on alanine aminotransferase (ALT) activity or lactate dehydrogenase (LDH) isozyme leakage in serum in mice 7 h after administration of rhTNF alone. Administration of rhTNF to GalN-sensitized mice resulted in marked increases in ALT activity and LDH isozyme leakage relative to those in mice treated with rhTNF alone. In mice pretreated with PMB, the levels of ALT and LDH isozyme leakage 7 h after rhTNF/GalN injection were significant decreased as compared with those in mice treated with rhTNF/GalN. Similarly, injection of PMB markedly decreased lipid peroxide formation in the liver of the GalN-sensitized mice treated with rhTNF. The injection of a low endotoxin dose (0.1 mg/kg, i.p.) markedly increased the lethality in mice treated with rhTNF (5x10(3) U/mouse, i.v.) and GalN, and these animals showed 100% mortality at 8 h. These findings suggested that the extent of TNF-induced toxicity caused by GalN administration may be a result of synergism between TNF and gut-derived endotoxin. It is likely that endogenously produced endotoxin play a significant role in rhTNF/GalN-hypersensitized mice.

Enhancement of doxorubicin activity in multidrug-resistant cells by mefloquine.

We studied the effect of the antimalarial drug mefloquine on the resistance of K562 cells to doxorubicin. Mefloquine synergistically potentiated the cytotoxicity of doxorubicin for doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 0.5-3 microM, but had hardly any synergistic effect in the parental cell line (K562) at the same concentration. Mefloquine was more potent than verapamil, a known modulator of multidrug-resistance. Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of mefloquine and of P-glycoprotein activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123. Our results indicate that mefloquine inhibits the P-glycoprotein pump-efflux activity in a dose-related manner. Moreover, mefloquine reduces the expression of the immunoreactive P-glycoprotein in K562/DXR cells as evaluated by cytofluorimetric assay. Taken together, the results indicate that mefloquine reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Reversal of multidrug resistance in human leukemia K562 by tamolarizine, a novel calcium antagonist.

A new type of organic Ca2+ channel blocker, tamolarizine, was examined for its reversing effect on multidrug-resistant tumor cells. Tamolarizine synergistically potentiated the cytotoxicity of doxorubicin for doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 0.1-10 microM, but had hardly any synergistic effects in the parental cell line (K562) at the same concentration. Moreover, tamolarizine inhibits the P-glycoprotein pump-efflux activity in a dose-related manner and reduces the expression of the immunoreactive P-glycoprotein in K562/DXR cells as evaluated by cytofluorimetric assay. These results indicate that tamolarizine reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Potentiation of acetaminophen hepatotoxicity by doxapram in mouse primary cultured hepatocytes.

The augmentation by doxapram (DOP) of the reduction in viability and of the apoptosis of cells induced by acetaminophen (AA) was examined in mouse primary cultured hepatocytes. Loss of viability on exposure to AA and/or DOP in cultured hepatocytes was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the apoptosis of cultured hepatocytes was detected by nuclear morphologic observation and from a ladder-like DNA fragmentation pattern. The combination of AA (5 mM) and DOP (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased the oxidative stress. Hepatocytes exposed for 24 h to AA (5 mM) plus DOP (100 microM), showed atrophy of nuclei, including chromatin condensation and a ladder-like DNA fragmentation pattern, characteristic of apoptosis. Benzyl-oxycarbonyl-Asp-CH2OC (O)-2,6-dichlorobenzene (Z-Asp-CH2-DCB, 50 microM), an inhibitor for caspases, improved the viability and ladder-like DNA fragmentation in cells exposed to DOP (200 or 500 microM) alone or AA ( 5 mM) plus DOP (100 microM). However, loss of viability on exposure to a high concentration of AA (10 mM) and ladder-like DNA fragmentation were not affected by Z-Asp-CH2-DCB. These results indicated that the synergistic increase in oxidative stress, activation of caspases and DNA fragmentation induced by DOP potentiated the hepatotoxicity of AA.

Characterization of hydrogen peroxide-induced apoptosis in mouse primary cultured hepatocytes.

The influence of oxidative stress by hydrogen peroxide (H2O2) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30 min in saline A containing H2O2. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H2O2. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addition of antioxidants (N-acetylcysteine, L-ascorbic acid), a metal-chelator (1,10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H2O2-induced cell viability reduction suggested that oxidative stress by H2O2 itself or H2O2-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.

Reversal of acquired resistance to doxorubicin in K562 human leukemia cells by astemizole.

This study demonstrates that astemizole, a non-sedating anti-histaminergic drug with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of doxorubicin in doxorubicin-resistant human leukemia cells (K562/DXR). Astemizole synergistically potentiated the cytotoxicity of doxorubicin for K562/DXR cells at a concentration of 0.1-3 microM in a dose-dependent manner, whereas they showed hardly any synthergistic effect in the parental cell line (K562) at the same concentration. Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of astemizole on P-glycoprotein activity in cytofluorographic efflux experiments with doxorubicin. Our results indicate that astemizole inhibits the P-glycoprotein pump-efflux activity in a dose-related manner. Moreover, it also inhibits the photolabeling of P-glycoprotein by [3H]azidopine in a dose-dependent manner. These findings provide a biological basis for the potential therapeutic application of astemizole as an anticancer drug either alone or in combination with doxorubicin to multidrug-resistant leukemic cells.

Role of endogenous endotoxin on tumor necrosis factor-hypersensitivity caused by D-galactosamine challenge.

We examined the role of endotoxin in the mechanism of recombinant human tumor necrosis factor (rhTNF)-hypersensitivity caused by D-galactosamine (GalN). We used polymyxin B, an antibiotic with anti-endotoxin activity, to determine the participation of endogenous endotoxin. The glycogen and blood glucose level of rhTNF (1 x 10(4) units/mouse, i.v.)-injected mice was lower at 7 h post-intoxication than that in the control. Administration of rhTNF to GalN (700 mg/kg, i.p.)-treated mice resulted in lower levels of glycogen and blood glucose than those in animals treated with rhTNF alone. In mice pretreated with polymxin B (20 mg/kg, i.p.), the level at 7 h after rhTNF/GalN-injection was markedly increased compared to that in mice treated with rhTNF/GalN alone. The injection of a low endotoxin dose (0.1 mg/kg, i.p.) markedly decreased the rectal temperature in mice treated with rhTNF (5 x 10(3) units/mouse, i.v.) and GalN, and none of these animals survived after treatment for 18 h. These findings suggest that endogenously produced endotoxin may contribute to the extent of rhTNF-hypersensitivity caused by GalN.

Roles of selenium in endotoxin-induced lipid peroxidation in the rats liver and in nitric oxide production in J774A.1 cells.

We examined the role of selenium (Se) in the mechanism of oxidative stress caused by endotoxin by feeding rats deficient a diet in this element. In rats fed the Se-deficient diet (concentration of Se, less than 0.027 microg g(-1)) for 10 weeks, Se level and glutathione peroxidase (GSH-Px) activity in the liver were about 47 and 43% lower, respectively, than those in rats fed a Se-adequate diet (Se, 0.2 microg g(-1)). Rat fed the Se-deficient diet and given endotoxin (6 mg kg(-1), i.p.) showed a mortality rates of about 43% at 18 h. Nevertheless, no lethality was observed with endotoxin (4 mg kg(-1), i.p.) challenge. Levels of serum lactate dehydrogenase and acid phosphatase leakage were significantly higher in Se-deficient rats than those in Se-adequate diet 18 h after endotoxin (4 mg kg(-1), i. p.) challenge. Superoxide anion generation and lipid peroxide formation in the liver of Se-deficient rat were markedly increased 18 h after endotoxin (4 mg kg(-1), i.p.) injection compared with those in the endotoxin/Se-adequate diet group, whereas non-protein sulfhydryl level in the liver after administration of endotoxin to Se-deficient rats was lower than that in Se-adequate rats treated with endotoxin. We investigated whether Se can suppress nitric oxide (NO) generation and cytotoxicity in endotoxin-treated J774A.1 cells. Treatment with Se (10(-6) M) markedly inhibited endotoxin (0.1 microg ml(-1))-induced NO production in J774A.1 cells. Se induced an increased activity of GSH-Px in cells after 24 h of incubation, suggesting that the preventive effect of Se on NO production in endotoxemia is due to the induction of Se-GSH-Px activity. However, Se did not affect endotoxin-induced cytotoxicity in J774A.1 cells. These findings suggested that the oxidative stress caused by endotoxin may be due, at least in part, to changes in Se regulation during endotoxemia.

Tandem repeat structure of rhamnose-binding lectin from catfish (Silurus asotus) eggs.

The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures.

Hot water extract of bark of Nikko maple (Acer nikoense) induces apoptosis in leukemia cells.

In screening for antitumor constituents in traditional crude drugs, we used three cultured cell lines: mouse leukemia P388 cells, doxorubicin-resistant P388 cells and leczyme (catalytic lectin)-resistant P388 cells. The hot water extract (HWE) of the bark of Nikko maple (Acer nikoense) showed concentration-dependent inhibitory effects on the growth of these three cell lines. DNA fragmentation and morphological changes, accompanied by condensed and fragmented nuclei, were observed in the leukemia cell lines cultured with HWE of the bark of Nikko maple. Treatment with this HWE increased the expression of sialylated glycoconjugates on the apoptotic cells. These results suggest that HWE induces cell death via apoptosis in vitro.

Thalidomide promotes the release of tumor necrosis factor-alpha (TNF-alpha) and lethality by lipopolysaccharide in mice.

We investigated the in vivo effects of thalidomide on the production of tumor necrosis factor-alpha (TNF-alpha). An in vivo systemic release of TNF-alpha occurred after the injection of lipopolysaccharide (LPS) in male ddY mice, and the TNF-alpha serum levels reached 652.2 +/- 75.7 pg/ml 90 min after the injection of LPS (0.3 mg/kg, i. p.). When thalidomide (1, 3, or 6 mg/kg) was administered intraperitoneally 3 h before the injection of LPS (0.3 mg/kg, i. p.), thalidomide markedly enhanced LPS-induced TNF-alpha release in a dose-dependent manner. The TNF-alpha serum levels at 90 min were 640 +/- 58.6, 1985 +/- 132.6, and 2795 +/- 203.5 pg/ml, respectively, compared to 628.6 +/- 64.4 pg/ml in mice treated with LPS-alone. Pretreatment with a single injection of thalidomide (1, 3, or 6 mg/kg, i. p.) dose-dependently increased the subsequent mortality caused by a challenge with LPS (15 mg/kg, i. p.), a dose that caused death in 10% of the control mice. We conclude that thalidomide enhances in vivo TNF-alpha secretion and the lethality of LPS in mice.

Cepharanthine inhibits proliferation of cancer cells by inducing apoptosis.

Cepharanthine, a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata was examined for a possible apoptosis-inducing effect in murine P388 doxorubicin-sensitive (P388/S) and -resistant (P388/DOX) cells. A significant increase in LDH release from cells was observed after P388/S and P388/DOX cells had been exposed to cepharanthine for 24 h. Cepharanthine (10 micrograms/ml) markedly induced apoptosis in resistant cells after 6 h and 24 h. By the means of agarose electrophoresis the DNA ladder was detected in cell lines treated with cepharanthine for 24 h. Cepharanthine (1-10 micrograms/ml) also induced the production of reactive oxygen species in P388/DOX cells, while no such cepharanthine-induced increase in reactive oxygen species was observed in P388/S cells. Flow cytometry analysis showed a high level of Fas-antigen expression in P388/DOX cells treated with cepharanthine. Furthermore, we found that the inhibitition of DNA and protein synthesis caused by cepharanthine (10 micrograms/ml) was more significant in resistant cells than in sensitive cells. Cepharanthine had no effect on the GSH content of P388/S and P388/DOX cells. Our experimental results suggest that cepharanthin can induce apoptosis both in P388/S and P388/DOX cells, especially in the latter. Apoptosis induced by cepharanthine may be implicated in the production of reactive oxygen species and Fas-antigen expression in tumor cells.

Apoptotic cell death induced by physarumin (hemagglutinin from myxomycete, Physarum polycephalum).

Physarumin, a carbohydrate-binding protein (hemagglutinin or lectin), was isolated from the plasmodium of Physarum polycephalum. Physarumin agglutinated not only several species of erythrocytes but also tumor cells such as AH109A ascites hepatoma cells, sarcoma 180 ascites cells and mouse leukemia P388 cell lines. Physarumin had tumor cell growth-inhibitory activity, and induced the apoptosis of P388 cell lines. Physarumin-induced apoptosis required binding to a 68 kDa counter-receptor on the P388 cell surface. Since the agglutinating and antiproliferative activities of physarumin were inhibited by asialofetuin and thyroglobulin, respectively, it is suggested that physarumin reacts with the galactose moiety of carbohydrate chains of physarumin receptor.

Potentiation of pirarubicin activity in multidrug resistant cells by rifampicin.

The effect of the anti-tuberculosis drug rifampicin on pirarubicin activity was investigated in multidrug-resistant cells overexpressing P-glycoprotein. Rifampicin increased the sensitivity of pirarubicin to anthracycline-resistant mouse leukemic P388 cells and significantly enhanced the cytotoxicity and intracellular accumulation of pirarubicin in resistant cells, but had no effect in parent cells. By contrast, two other rifamycins, rifamycin B and SV, had no effect on pirarubicin accumulation in resistant cells. Rifampicin also enhanced pirarubicin-induced apoptosis and G2/M blockade on the cell cycle in resistant cells. These results show that rifampicin enhances the cytotoxic action of pirarubicin in resistant cells, at least partly via the inhibition of cellular pirarubicin efflux.

Reversal of multidrug resistance by tacrolimus hydrate.

Tacrolimus hydrate, a potent immunosuppressant produced by Streptomyces tsukubaensis, was examined for its effect on epirubicin activity in multidrug-resistant P388 leukemia (P388/R) cells overexpressing P-glycoprotein and the parent (P388/S) cells. In the absence of modulator, the 50% inhibitory concentration for epirubicin after 48-h incubation, determined using a microculture tetrazolium assay, was 0.8 microgram/ml in P388/R cells and 0.009 microgram/ml in P388/S cells. P388/R cells demonstrated a 90-fold reduction in sensitivity to epirubicin. Tacrolimus hydrate (1 and 10 microM) markedly enhanced epirubicin cytotoxicity by 4.2- and 26.7-fold for P388/R cells. A significant increase in LDH release from cells by tacrolimus hydrate was also observed in P388/R cells treated with epirubicin. Tacrolimus hydrate had a marked effect on epirubicin-induced G2/M blockade in the resistant cells. Both tacrolimus hydrate and cyclosporin A dramatically increased the accumulation of epirubicin by the resistant cells, while these compounds had no effect on epirubicin accumulation in the parent cells. Thus, tacrolimus hydrate is able to down-modulate P-glycoprotein-associated resistance through inhibition of P-glycoprotein function, suggesting that the drug may be a candidate for killing drug-resistant tumor cells.

The enhancing effect of tumour necrosis factor-alpha on oxidative stress in endotoxemia.

The enhancing effect of tumour necrosis factor-alpha (TNF-alpha) on oxidative stress with or without a sublethal dose of endotoxin was examined. The mortality of mice treated with recombinant human TNF-alpha (1 x 10(4) units/mouse, intravenously) and endotoxin (0.01-1 mg/kg, intraperitoneally) was dependent on the dose of endotoxin. The liver lipid peroxide level, superoxide anion generation and serum lactate dehydrogenase activity, especially serum lactate dehydrogenase-5 isozyme leakage, in mice 2-4 hr after administration of recombinant human TNF to endotoxin-pretreated mice (0.5 mg/kg, intraperitoneally) were markedly higher than in those without endotoxin, whereas the administration of recombinant human TNF significantly decreased the non-protein sulfhydryl level, superoxide dismutase and glutathione peroxide activities in the liver of endotoxin-injected mice compared with those in mice treated with recombinant human TNF or endotoxin alone. Furthermore, findings clearly demonstrated that J774A.1 cells stimulated with recombinant human TNF (1 x 10(4) units/ml) can effectively produce nitric oxide in the presence of endotoxin, and the production was dependent on the dose of endotoxin (0.01-10 micrograms/ml). The level of lipid peroxide in mice 4 hr after administration of recombinant human TNF and lead acetate (50 mg/kg, intravenously) was markedly higher than that in the mice treated with recombinant human TNF alone. By contrast, injection of polymyxin-B (20 mg/kg, intraperitoneally, an anti-endotoxin drug) markedly decreased the lipid peroxide level in the liver of the mice treated with recombinant human TNF and lead acetate. These findings suggest that the oxidative stress caused by TNF occurs as a enhancing effect of endotoxin or by bacterial translocation from the intestinal gut under reduction of reticuloendothelial system function in various disease states, and that the effect of TNF may cause a marked increase of toxicity of oxidative stress by endotoxin.