Induction of metallothionein by cadmium-metallothionein in rat liver: a proposed mechanism.

Distribution of Cd to various organs following iv administration of CdCl2 (3.5 mg Cd/kg) resulted in more than 43% of total tissue Cd accumulating in the liver. In contrast, after CdMT administration (0.5 mg Cd/kg), only 1% of the Cd was found in liver. Rats administered CdCl2 (1.0 mg Cd/kg) had hepatic MT values 30-fold greater than controls and a hepatic Cd concentration of 17 micrograms/g. In comparison, rats treated with CdMT (0.4 mg Cd/kg) had hepatic MT concentrations 7-fold greater than controls and a hepatic Cd concentration of 0.80 micrograms/g. However, when hepatic MT levels were normalized to tissue Cd concentrations, induction of MT by CdMT was 5-fold greater than by CdCl2. Northern and slot-blot analyses of mRNA showed that both CdCl2 and CdMT coordinately increased MT mRNA. These data suggest that both CdMT and CdCl2 increase hepatic MT by similar mechanisms. A dose-response increase in MT produced by CdCl2 indicated a biphasic response, with low doses producing relatively more hepatic MT than higher doses. In addition, the amount of MT produced per unit Cd after CdMT treatment was similar to those observed after low doses of CdCl2 in the dose-response experiment. These data provide strong evidence to support the conclusion that the apparent potency of CdMT observed here and in previous studies is most likely due to the small amount of Cd distributed to the liver, which is relatively more effective in inducing MT than are higher concentrations.     

Toxicological evaluation of ammonium 4,8-dioxa-3H-perfluorononanoate,a new emulsifier to replace ammonium perfluorooctanoate in fluoropolymer manufacturing

Ammonium 4,8-dioxa-3H-perfluorononanoate (ADONA) was developed to replace ammonium perfluorooctanoate (APFO) as an emulsifier in the manufacture of fluoropolymers. The toxicity of ADONA was evaluated in acute and repeat-dose studies of up to 90-days duration, and in eye and skin irritation, dermal sensitization, genotoxicity, and developmental toxicity studies. ADONA was also evaluated as a peroxisome proliferator-activated receptor alpha (PPARα) agonist in rats. ADONA was moderately toxic orally and practically non-toxic dermally in acute studies in rats. It was a mild skin irritant and a moderate to severe eye irritant in rabbits. It was a weak dermal sensitizer in local lymph node assays in mice. ADONA was not genotoxic based on the weight of evidence from five assays. It was not developmentally toxic in rats except at maternally toxic doses. ADONA was a possible PPARα agonist in male rats. The liver was the primary target organ in male rats and the kidney was the primary target organ in female rats. NOAELs in 28- and 90-day oral studies in rats were 10 mg/kg/day for males and 100 mg/kg/day for females. These findings demonstrate that the toxicity profile for ADONA is acceptable for its intended use and is superior to that of APFO.     

受体树突状细胞诱导供体特异性的免疫低反应及其可能机制

目的探讨受体耐受性树突状细胞(DC)在诱导供体特异性免疫耐受中的作用及其可能机制。方法通过以B7反义肽封闭的负载供体抗原的受体DC对受体小鼠进行预处理,3d或2个月取脾脏分离T细胞,与负载供体抗原或无关供体抗原的DC作混合淋巴细胞反应,并且在3d或2个月时以负载供体抗原的受体DC对受体小鼠进行再次免疫后,RT-PCR半定量法测定脾脏中细胞因子mRNA表达。结果以B7反义肽封闭的负载供体抗原的受体DC预处理3d或2个月后的受体T细胞,产生对间接途径提呈的供体抗原的免疫低反应;同时脾脏IL-10表达升高,而IL-2、IFN-γ表达降低。结论 B7反义肽封闭的受体DC可能通过免疫偏移诱导针对间接途径提呈的供体抗原特异性的免疫低反应。     

Toxicity of ammonium perfluorooctanoate in male cynomolgus monkeys after oral dosing for 6 months.

Ammonium perfluorooctanoate (APFO) is a processing aid in the production of fluoropolymers that has been shown to have a long half-life in human blood. To understand the potential toxicological response of primates, groups of male cynomolgus monkeys were given daily po (capsule) doses of either 0, 3, 10, or 30 (reduced to 20) mg/kg/day for 26 weeks. Two monkeys from each of the control and 10 mg/kg/day dose groups were observed for 90 days after the last dose. Clinical observations, clinical chemistry, determination of key hormones, gross and microscopic pathology, cell proliferation, peroxisomal proliferation, bile-acid determination, and serum and liver perfluorooctanoate (PFOA) concentrations were monitored. Toxicity, including weight loss and reduced food consumption, was noted early in the study at the 30 mg/kg/day dose; therefore, the dose was reduced to 20 mg/kg/day. The same signs of toxicity developed in 3 monkeys at 20 mg/kg/day, after which treatment of these monkeys was discontinued. One 30/20 mg/kg/day monkey developed the signs of toxicity noted above and a possible dosing injury, and this monkey was sacrificed in extremis on Day 29. A 3 mg/kg/day dose-group monkey was sacrificed in extremis on Day 137 for reasons not clearly related to APFO treatment. Dose-dependent increases in liver weight as a result of mitochondrial proliferation occurred in all APFO-treated groups. Histopathologic evidence of liver injury was not observed at either 3 or 10 mg/kg/day. Evidence of liver damage was seen in the monkey sacrificed in moribund condition at the highest dose. Body weights were decreased at 30/20 mg/kg. PFOA concentrations in serum and liver were highly variable, were not linearly proportional to dose, and cleared to background levels within 90 days after the last dose. A no observable effect level was not established in this study, and the low dose of 3 mg/kg/day was considered the lowest observable effect level based on increased liver weight and uncertainty as to the etiology leading to the moribund sacrifice of one low-dose monkey on Day 137. Other than those noted above, there were no APFO-related macroscopic or microscopic changes, changes in clinical chemistry, hormones, or urinalysis, or hematological effects. In particular, effects that have been associated with the development of pancreatic and testicular toxicity in rats were not observed in this study.     

Induction of NAD(P)H:quinone reductase by probucol: a possible mechanism for protection against chemical carcinogenesis and toxicity

Dietary antioxidants protect laboratory animals against induction of tumours by a variety of chemical carcinogens. Among possible mechanism, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes, including quinone reductase and glutathione S-transferase (GSH transferase). Probucol is used cholesterol-lowering drug used in the clinic, with pronounced antioxidant effect that protect against chemical carcinogenesis and toxicity. In the present study we therefore examined the ability of probucol to induce activities of quinone reductase in the cytosolic fractions of various tissues of mice. Quinone reductase activity was increased significantly in 6 of 8 tissues examined from probucol-fed mice. The greatest proportionate increase, to 1.8 times control levels, was observed in liver. Probucol also increased quinone reductase activities of forestomach, heart, kidney, lungs and spleen. Quinone reductase is a major enzyme of xenobiotic metabolism that carries out obligatory two-electron reductions and thereby protects cells against toxicity of quinones. It is induced in many tissues coordinately with other enzymes that protect against electrophilic toxicity. The protective effects of probucol appear to be due, at least in part, to the ability of this antioxidant to increase the activities in rodent tissues of several enzymes involved in the non-oxidative metabolism of a wide variety of xenobiotics. The induction of such enzyme, quinone reductase by probucol suggests the potential value of this compound as a protective agent against chemical carcinogenesis and other forms of electrophilic toxicity. The significance of these results can be implicated in relation to cancer chemopreventive effects of probucol in various target organs.     

Antiplatelet effect of amlodipine: a possible mechanism through a nitric oxide-mediated process.

The effect of amlodipine, a novel calcium channel blocker of the dihydropyridine type, on rabbit platelet aggregation, and the possible antiaggregatory mechanisms of amlodipine, especially on the nitric oxide (NO) guanosine 3',5'-cyclic monophosphate (cyclic GMP)-mediated pathway, were investigated. Other effects of amlodipine on thromboxane B2 (TXB2) formation in platelets also were examined. Amlodipine concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/mL) or thrombin (0.1 U/mL) with an IC50 range of 32-69 microM. Along with this inhibition, our results also demonstrated that in the presence of L-arginine (100 IM), amlodipine (50 microM) increased nitric oxide synthetase (NOS) activity (from the resting activity of 2.05+/-0.36 to 7.11+/-0.95 pmol/mg protein/min) and NO release (by 80%), accompanied by an elevation of the cyclic GMP level (from the resting platelet level of 1.27+/-0.12 to 6.21+/-0.55 pmol/10(9) platelets) induced by collagen (10 microg/mL). However, the antiaggregatory effect of amlodipine (50 microM) could be attenuated significantly by oxyhemoglobin (5 microM), a NO scavenger, or N(G)-nitro-L-arginine methyl ester (100 microM), a specific NOS inhibitor. In addition, the TXB2 production in platelets induced by collagen or thrombin was concentration-dependently inhibited by amlodipine. Therefore, we propose that the antiaggregatory mechanisms of amlodipine might be mediated, in part, by a NO-cyclic GMP process accompanied by the inhibition of TXB2 formation in platelets.     

Mechanism for Species-Specific Induction of Leydig Cell Tumors in Rats by Lansoprazole

Lansoprazole is a substituted benzimidazole which inhibits gastricacid secretion by inhibiting the hydrogen-potassium ATPase (protonpump) in the parietal cell. The finding of Leydig cell hyperplasiaand Leydig cell tumors in 2-year oral studies in Sprague-Dawleyrats but not in CD-1 mice prompted investigative studies todetermine the mechanism for the Leydig cell changes. hCG challengestudies in Sprague-Dawley rats revealed decreased testosteroneresponsiveness in rats treated orally for 1 or 2 weeks withlansoprazole. After 4 weeks of daily oral treatment increasesin serum LH and decreases in serum testosterone were detectedwithin a few hours after dosing. In a study where 9-month-oldmale F344 rats were given testosterone supplementation via Silasticimplants and then treated with lansoprazole for 6 months, ahigh incidence of Leydig cell tumors was seen in lansoprazoletreated,unsupplemented rats, whereas no Leydig cell tumors were seenin testosterone supplemented rats. This implied that reductionof the normal feedback inhibition at the level of the hypothalaumsand/or pituitary due to reduced testosterone levels, thus givingrise to elevated levels of LH, was involved in the inductionof Leydig cell tumors by lansoprazole. In vitro studies withLeydig cells from rats using various stimulators and precursorsof testosterone biosynthesis demonstrated that the most sensitivesite for inhibition of testosterone synthesis by lansoprazoleis the transport of cholesterol to the cholesterol side chaincleavage enzyme. The IC50s for inhibition of LH or hCG-stimulatedtestosterone synthesis in Leydig cells from rats, mice, andmonkeys were 11–12, 8, and 27.4 µg/ml, respectively.In vitro studies with metabolites of lansoprazole revealed thatthree metabolites were more potent inhibitors of testosteronesynthesis than the parent drug, two of them being at least 10times more potent. These metabolites are present in rats atsubstantial levels but are undetectable in humans. The lackof induction of Leydig cell tumors in mice, lower sensitivityof primate Leydig cells, and the absence of testosterone synthesisinhibitingmetabolites in man suggest that Leydig cell tumors found inrats represent a species-specific sensitivity and does not implya risk for clinical use in man.     

A possible mechanism of infertility by acute cadmium intoxication.

A possible mechanism of infertility by Cd was investigated from the standpoint of influence of Cd on the contractile responses of isolated seminal vesicle from guinea pigs to K, acetylcholine, noradrenaline, Ba and Ca by using the Magnus method. Cd inhibited the contractile responses to the contractile agents tested in a dose-dependent manner. Cd showed the preferential inhibition against the contractile responses to K and Ca. The inhibitory effect of Cd on the contractile responses to noradrenaline and acetylcholine was hardly removed. The effect of Cd was inhibited by the thiol compounds, glutathione and thiola. The active mechanism of Cd was discussed in relation to Ca mobilization.     

Induction of peroxisomes by treatment with perfluorooctanoate does not increase rates of H2O2 production in intact liver.

Increases in acyl coenzyme A (CoA) oxidase activity due to peroxisome proliferation are postulated to cause oxidative stress via elevated production of H2O2, leading to DNA damage. These changes are suspected to be responsible for tumor formation caused by non-genotoxic carcinogens which do not bind to DNA but cause proliferation of peroxisomes. However, the activity of the peroxisomal enzyme acyl CoA oxidase assayed in vitro in the presence of excess fatty acyl CoA substrate may not reflect rates of H2O2 generation in intact liver where fatty acid supply is carefully controlled in part by delivery of substrate. The purpose of this work was to determine if rates of hepatic H2O2 generation were altered in perfused liver and in vivo following induction of H2O2-generating acyl CoA oxidase activity. Injection of the potent peroxisome proliferating agent perfluorooctanoate into rats 5 days prior to sacrifice caused an expected 4-fold increase of H2O2-generating acyl CoA oxidase activity measured in hepatic homogenates. In contrast, rates of H2O2 generation in perfused liver measured spectrophotometrically (660-640 nm) through a lobe of the liver were not altered by perfluorooctanoate treatment (7.3 +/- 1.5 vs. 7.8 +/- 0.5 mumol/g/h in livers from untreated control rats). Similar treatment with perfluorooctanoate also increased in vitro acyl CoA oxidase activity 9-fold in livers from deermice; however, rates of elimination of methanol, a selective substrate for catalase in rodents whose oxidation is limited by the supply of H2O2, were not altered significantly in vivo (control, 110 +/- 11 mumol/g/h vs. perfluorooctanoate, 112 +/- 32 mumol/g/h). Taken together, these data demonstrate that elevation of H2O2 formation by acyl CoA oxidase activity measured in vitro is not necessarily associated with increases in rates of H2O2 generation in intact perfused liver or in vivo, most likely due to rate-limitation in intact cells by fatty acid supply. These data do not support the hypothesis that the induction of peroxisomes leads to excessive H2O2 production and oxidative stress. It follows that alternative hypotheses to explain carcinogenesis caused by peroxisome-proliferating agents need to be considered.     

Hepatocellular carcinoma caused by iron overload: a possible mechanism of direct hepatocarcinogenicity

BACKGROUND/AIMS: Excess hepatic iron may be both directly and indirectly carcinogenic. The aim of this study was to determine if generation of reactive oxygen species and the resulting oxidative damage induced by free hepatic iron is directly hepatocarcinogenic. METHODS: Sixty male Wistar albino rats were iron-loaded by ferrocene supplementation of their diet. Biochemical parameters of oxidative damage and lipid peroxidation, DNA unwinding and strand breaks, and the Ames Mutagenesis Test were measured at 4 monthly intervals and correlated with the degree of hepatic iron overload, the presence of iron-free preneoplastic foci in the liver, and the development of hepatocellular carcinoma in comparison with 60 control rats. RESULTS: Levels of lipid hydroperoxides, malonaldehyde, 8-isoprostane and 8-hydroxy-2'-deoxyguanosine increased, reaching peak concentrations at 20-24 months, and correlating with an increase in the rate of DNA unwinding, strand breaks, and positive Ames Tests. Iron-free neoplastic foci became evident at 16 months and thereafter increased in number. Preneoplastic foci were present in five of eight rats remaining at 32 months and HCC had developed in one of the five. CONCLUSIONS: Our findings are compatible with the hypothesis that the direct hepatocarcinogenic effect of free iron is mediated by the generation of oxygen reactive species and oxidative damage that are mutagenic and carcinogenic.     

大花紫玉盘素诱导肿瘤多药抗药性细胞凋亡及其机制

目的比较研究番荔枝内酯大花紫玉盘素(uvarigrin)诱导多药抗药性KBv200细胞及其亲本KB细胞凋亡及其机制。方法以MTT法进行细胞毒测定；用Annexin V FITC染色及流式细胞仪检测细胞凋亡。活性氧(ROS)测定以DCFH-DA标记，细胞线粒体跨膜电位(ΔΨ_m)测定用DiOC₆标记，均以流式细胞仪检测。Caspase-9激活的测定用Western blotting法。结果大花紫玉盘素对KBv200细胞及其亲本KB细胞的生长均有明显的抑制作用；大花紫玉盘素不仅能介导KB细胞凋亡，而且也能介导KBv200细胞凋亡；大花紫玉盘素作用于KBv200细胞及其亲本KB细胞12，24和48 h，均引起ROS升高以及ΔΨm降低，而且呈时间依赖性。Western blotting方法分析显示Caspase-9被激活。结论大花紫玉盘素可能通过线粒体通路诱导细胞凋亡。     

Lipid peroxidation: a possible mechanism of trichloroethylene-induced nephrotoxicity

The purpose of this study was to investigate whether lipid peroxidation plays a role in (TCE) trichloroethylene-induced nephrotoxicity in mice at different oxygen concentrations. Male NMRI mice (25-30 g) were treated i.p. with TCE in a dosage of 125-1000 mg/kg in sesame oil. To determine the TCE-induced depletion of reduced glutathione (GSH) in the kidney cortex and liver tissue, mice were given 1000 mg/kg TCE i.p., then killed between 0 and 6 h after TCE administration and GSH was measured was non-protein sulfhydryls. In another series of experiments, mice were administered 125 to 1000 mg/kg TCE i.p. with or without a 2 h i.p. pretreatment with 1500 mg/kg L-buthionine-S-R-sulfoximine (BSO). Mice were then exposed to a 10, 15, 20 or 100% oxygen atmosphere for 3 h and lipid peroxidation in vivo was measured as exhalation of ethane. Subsequently, mice were killed and malondialdehyde (MDA) generation was measured in the liver and kidney cortex. Ethane evolution was estimated by gas chromatography and MDA was determined as thiobarbituric acid reactive substances. In a further series of experiments mice were treated in the same manner as for ethane and MDA determination and the changes in blood urea nitrogen (BUN) and accumulation of the organic ion p-aminohippurate (PAH) were determined. PAH accumulation by renal cortical slices were measured as the slice to medium (S/M) ratio. Six hours after administration of 1000 mg/kg TCE to mice, GSH was significantly depleted to about 60% of control in the kidney cortex but not in the liver. Three hours after TCE administration, MDA content in the kidney cortex and ethane exhalation increased in a dose-dependent manner only under a 10% oxygen atmosphere. Under the same experimental conditions, MDA content remained unchanged in the liver. BSO depletion of GSH prior TCE administration induced an increase of the MDA content in the kidney cortex and an increase of the ethane exhalation in vivo. At 10% oxygen concentration, TCE induced a dose-dependent increase in BUN and a dose-dependent decrease of PAH accumulation by the renal cortical slices. Thus, the results of the present study suggest that, under hypoxic conditions, lipid peroxidation plays a role in TCE nephrotoxicity.     

Inhibition of thymidylate synthetase of walker carcinoma by chlorambucil. a possible mechanism of action

Effects of chlorambucil (CA) on growth of Walker carcinoma 256 and on enzymes involved in the synthesis of thymidine monophosphate are described. A single intraperitoneal dose (25 $\text{mg}\text{kg}$) or multiple doses (8mg/kg/day for 3 days) of CA were “curative” for drug-sensitive (WS) tumors. Thymidylate (dTMP) synthetase activity of WS tumors was significantly decreased (approximately 25%) at 3 hr, reached its lowest level (approximately 50% loss) at 12 hr. and remained at this level up to 60 hr after administration of 25 $\text{mg}\text{kg}$ of CA. Dihydrofolate (FAH₂) rcductase activity did not change significantly up to 36 hr and then slowly decreased (approximately 20% loss) by 60 hr. Thymidine (TdR) kinase activity of WS tumors was not affected. Treatment of WS tumors with multiple doses of CA also resulted in pronounced inhibition of dTMP synthetase activity (approximately 50% loss). some decrease (approximately 15% loss) in FAH₂ reductasc activity and no change in TdR kinase activity. A Chlorambucil-resistant strain (WR) of Walker carcinoma was developed. In contrast to WS, after treatment with CA the enzyme activities of WR tumors remained essentially the same as those from untreated animals. In vitro incubation of partially purified dTMP synthetase enzymes from either WS or WR tumors with CA inhibited both enzymes to the same extent (approximately 50% loss at 1.25 × 10^?6 M). TdR kinase and FAH₂ reductasc activities were not inhibited up to 1.25 × 10^?4 M CA. The results of these studies support the concept that CA exerts cytotoxic activity by inhibition of dTMP synthetase.Activities of dTMP synthetase and TdR kinase were found to be significantly altered in WR tumors as compared to WS tumors. The activity of dTMP synthetase was decreased approximately 20% and that of TdR kinase was increased approximately 35% in WR tumors. Resistance to CA may be due. in part, to increased dependence of WR tumors on the salvage pathway for synthesis of dTMP.     

Cocaine-induced hepatotoxicity: lipid peroxidation as a possible mechanism

In vitro experiments with hepatic washed microsomal preparations showed that malondialdehyde (MDA) formation was increased in a time- and concentration-dependent manner using COC or NC as the substrate. Though 1 mM COC or NC inhibited MDA formation, significant elevations were observed for 100, 10 or 1 microM concentrations. NC at 10 microM after a 30 minute incubation produced a 34% decrease in hepatic microsomal cytochrome P450 whereas 1 mM NC had no such effect. MDA formation in vivo, measured as total absorbance at 535 nm per gram liver, was found to be maximal 4 hours after 40 mg/kg NC ip. Elevations of serum transaminase (SGPT) however were not found until 6 hours after NC. We conclude from these studies that COC and NC induce lipid peroxidation in the liver of PB-pretreated Swiss-origin mice and that peroxidative attack may be a mechanism for hepatotoxicity of these compounds.     

Induction of apoptosis by 25-hydroxycholesterol in adult rat Leydig cells: protective effect of 17beta-estradiol

Testicular macrophages can convert cholesterol into 25-hydroxycholesterol which strongly stimulates Leydig cell testosterone production. We demonstrated that 25-hydroxycholesterol reduced cholesterol biosynthesis in adult rat Leydig cells. This oxysterol can also be cytotoxic. As hydroxylated cholesterol can induce apoptosis in various cells, we investigated cell death produced by 25-hydroxycholesterol. Apoptosis was characterized by TUNEL assay and by DAPI test. Addition of 25-hydroxycholesterol, during 24 h, induced a dose dependent increase of apoptosis. This effect was reduced by a treatment with a caspase-3 inhibitor (Ac-DEVD-CHO). 25-Hydroxycholesterol is known to stimulate testosterone production, but an increase of intracellular or culture medium testosterone level does not modify significantly the percentage of apoptotic cells. In contrast, addition of 17β-estradiol (2 nM) induced a decrease of apoptotic cells. These data suggested that this oxysterol can be used by rat Leydig cells in culture for sterol metabolism, but also induces apoptosis which could be inhibited by 17β-estradiol.     

Cocaine-induced respiratory depression in urethane-anesthetized rats: a possible mechanism of cocaine-induced death.

Urethane-anesthetized rats were used to study the mechanism of cocaine-induced death. Continuous recording of the changes in five physiological parameters, including respiratory rate (RR), electroencephalogram (EEG), blood pressure (BP), electrocardiogram (ECG), and body temperature (BT), were conducted after intraperitoneal (IP) administration of a single dose of cocaine HCl (70 mg/kg). In the control group (normothermic with core body temperature 37.7 +/- 0.1 degree C and spontaneously breathing), the death rate was 88% (15/17), and the average time to respiratory arrest was 12.99 +/- 1.40 min (mean +/- SEM). The first set of experiments investigated the contribution of hypothermia to cocaine-induced death. The hypothermic group (core body temperature 33.9 +/- 0.3 degrees C and spontaneously breathing) had a death rate of 81.5% (22/27), and an average time to respiratory arrest of 16.70 +/- 1.24 min, which was significantly (p les than 0.05) prolonged. A substantial decrease in respiratory rate was seen in normothermic group, while all the other measured parameters remained relatively stable until respiratory arrest. Sequential arterial blood gas data in this group showed a decrease in PaO2 from 116.0 +/- 5.7 mmHg to 57.7 +/- 4.6 mmHg, an increase in PaCO2 from 27.7 +/- 2.2 mmHg to 42.7 +/- 3.0 mmHg, and a decrease in pH from 7.467 +/- 0.039 to 7.357 +/- 0.003. To confirm that respiratory depression was an important mechanism of cocaine-induced death in this model, ten normothermic rats underwent mechanical ventilation, and all survived cocaine exposure. This study points to the important role of respiratory depression as a cause of cocaine-induced death.