Metallothionein: analysis in tissues and toxicological significance.

The chemical forms of cadmium in tissues can be largely divided into two groups, namely metallothionein (MT)-bound and non-MT-bound. Cadmium accumulates in the body mainly as the former, while the latter, the toxic form of cadmium, can be detected typically in tissues in two cases: (i) before induction of sufficient amounts of MT to sequester cadmium; and (ii) after the accumulation of cadmium in amounts such that it is beyond the capacity of the organ concerned to synthesize enough MT. We identified alcohol dehydrogenase as the major cadmium-binding protein in the liver before induction of MT, though the enzyme activity was not affected by such binding in vivo. The capacity to synthesize MT can be determined from the relative ratio of cadmium to co-existing metals (zinc and copper) in MT, and also by the increase in cadmium in the non-MT-bound form that follows the increase in this ratio. Non-MT-bound cadmium can be detected selectively by a histochemical method using a chelating agent, thiazolylazonaphthol.     

Cadmium carcinogenesis in male Wistar [Crl:(WI)BR] rats: dose-response analysis of effects of zinc on tumor induction in the prostate, in the testes, and at the injection site

The ability of zinc acetate to modify the carcinogenic effects of CdCl2 in male Wistar [Crl:(WI)BR] rats was studied over a 2-year period. Groups of rats received a single s.c. injection of Cd (30.0 mumol/kg) in the dorsal thoracic midline or i.m. in the right thigh at time 0. Zinc was given in three separate s.c. doses of 0.1, 0.3, or 1.0 mmol/kg (at -6, 0, and +18 h relative to cadmium) in the lumbosacral area or p.o. at 100 ppm in the drinking water (-2 to +100 weeks). Cadmium treatments (s.c.) resulted in the appearance of tumors at the injection site and in the testes. The incidence of s.c. injection site tumors (mostly mixed sarcomas) was markedly reduced by high dose (1.0 mmol/kg) s.c. zinc (50% reduction) and was almost abolished by p.o. zinc (92% reduction). Testicular tumors (mostly Leydig cell adenomas) induced by s.c. cadmium were reduced in a dose-related fashion by zinc and were found to be highly dependent on the ability of zinc to prevent the chronic degenerative effects of cadmium in the testes. Oral zinc had no effect on s.c. cadmium-induced testicular tumors, while i.m. cadmium alone did not induce these tumors. In rats in which s.c. cadmium-induced testicular tumors and chronic degenerative effects were prevented by zinc (1.0 mmol/kg, s.c.), a marked elevation in prostatic tumors (exclusively adenomas) occurred (control, 9.6%; cadmium plus high zinc 29.6%). Cadmium given i.m., which did not result in testicular tumors or degeneration, also induced an elevated incidence (42.3%) of prostatic tumors, again indicating a dependence on testicular function. Prostatic tumor incidence was also significantly elevated (25.0%) in rats receiving 1.0 mmol/kg zinc, s.c., in combination with i.m. cadmium. These results indicate that zinc inhibition of cadmium carcinogenesis is a complex phenomenon, depending not only on dose and route but also on the target site in question.     

Differences in chromatographic behaviour of rat and pig metallothionein isoforms: a possible method of distinguishing between exogenous and endogenous metallothioneins.

Part of the exogenous metallothionein present, e.g., in food of animal origin, can survive gastrointestinal digestion, and will be selectively taken up in the kidneys. The present paper describes a possible method of distinguishing exogenous cadmium metallothionein (CdMT) from its endogenous counterpart. Rat and pig metallothionein were purified from liver and kidneys of animals previously injected with cadmium chloride. In rats cadmium was present in two MT isoforms, RMT-1 and RMT-2. Ion-exchange chromatography of porcine liver cytosol also showed that two isoforms existed, but a major portion of the cadmium elutes with the second isoform, VMT-2. Using a reversed-phase HPLC system, the purified rat metallothionein isoforms eluted as single peaks before pig MT isoform VMT-2. The difference in chromatographic behaviour on reversed-phase HPLC between species-specific metallothioneins offers a unique possibility to study the fate of endogenous and exogenous metallothioneins simultaneously. This will be illustrated by the redistribution of 109Cd from exogenous CdMT to endogenous CdMT after intravenous injection of VMT-2 into rats.     

Experimental basis for increasing the therapeutic index ofcis-diamminedicarboxylatocyclobutaneplatinum(II) in brain tumor therapy by a high-zinc diet

Metallothionein (MT), a ubiquitous intracellular protein, confers resistance to the toxic effects of platinum compounds. Since a high-zinc diet has been shown to induce MT synthesis in extracerebral tissues but not in brain, we investigated whether it could provide an experimental basis for decreasing the hematotoxicity of carboplatin without impairing its activity against brain tumors. After 2 weeks on either a high-zinc diet or a control diet (zinc content, 180 vs 10 ppm), mice and rats received various doses of carboplatin or Hanks' balanced salt solution by i.p. injection. The hematotoxicity of carboplatin was evaluated with an assay of colony-forming units of granulocytes and mononuclear cells in mice. The high-zinc diet enabled a 50% increase in the carboplatin dose without increasing hematotoxicity. The antitumor activity was evaluated with an assay of the colony-forming efficiency of gliosarcoma cells from 9L brain tumors in rats. The high-zinc diet did not alter the efficacy of carboplatin against this brain tumor. Northern blot analysis confirmed that the high-zinc diet induced MT mRNA in the kidney but not in the brain of mice and rats; it also showed MT mRNA induction in bone marrow cells of mice but not in rat 9L brain tumors. These results suggest that increasing the dietary intake of zinc might increase the therapeutic index of carboplatin in the treatment of brain tumors.     

The high hepatocarcinogen susceptibility of LEC rats is genetically independent of abnormal copper accumulation in the liver

We previously reported that LEC rats, which show a spontaneousoccurrence of liver injury and hepatocellular carcinoma (HCC),are highly susceptible to chemical carcinogens such as diethylnitrosamine(DEN). Since abnormal copper accumulation in the liver of LECrats was found to be a cause of liver injury, it is necessaryto elucidate whether the carcinogen susceptibility of LEC ratsis related to the accumulation of copper in the liver. In thisstudy we have examined the relationship between the susceptibilityof F1 [LEC x LEA or LEC x Fischer 344 (F344)] and F1 backcrossrats to DEN and hepatic copper concentration, as copper accumulationhas been demonstrated to be inherited as an autosomal recessivetrait. The groups of F1 and F1 backcross rats were given a singleintraperitoneal injection of DEN (20 mg/kg body wt) and subjectedto a modified Solt—Farber protocol for assaying glutathioneS-transferase placental form (GST-P)-positive foci. The hepaticcopper concentration was examined by atomic absorption. Althoughno F1 rats showed a high copper concentration in the liver,the numbers of foci were as high as those in LEC rats whichaccumulate copper. Backcross rats separated into high and lowcopper concentration groups at an almost 1: 1 ratio, but therewas no significant difference in the mean numbers of foci betweenthese two groups. The results clearly indicate that the highsusceptibility of LEC rats to DEN is genetically independentof copper accumulation in the liver. A possible dominant inheritanceof this high carcinogen susceptibility was suggested. Biochemicalmeasurement of cytochromes P450 and b5 in the liver of F1 ratsindicated that alterations in drug metabolizing enzymes maybe partially responsible for the high carcinogen susceptibilityof LEC rats.     

Endogenous intestinal metallothionein possibly contributes to the renal accumulation of cadmium.

At low levels of dietary cadmium chloride, cadmium accumulates directly in the kidneys and not in the liver. As dietary cadmium induces intestinal metallothionein (MT), intestinal CdMT complexes could be at least partly responsible for the renal accumulation of oral cadmium. For this to be possible, however, serosal release of mucosal CdMT would be required. To test this hypothesis, we perfused isolated rat small intestinal segments (Fisher-Parsons method) in an attempt to demonstrate the release of intestinal MT. After two weeks of feeding dietary cadmium chloride, intestinal MT was induced in amounts proportional to the dietary cadmium level. Subsequent in vitro perfusion of the small intestine revealed a concentration-dependent release of intestinal MT on the serosal side. When 109CdCl2 was present in the perfusion medium, 109Cd appeared on the serosal side mainly in the MT fraction. These results indicate that endogenous intestinal MT may deliver CdMT to the organism, thus possibly contributing to the renal accumulation of orally ingested cadmium.     

Induction of 8-hydroxydeoxyguanosine but not initiation of carcinogenesis by redox enzyme modulations with or without menadione in rat liver.

Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.     

Trace element concentrations in renal cell carcinoma.

Cadmium, zinc, copper levels and zinc-copper, zinc-bromine, iorn-zinc, iron-copper and iron-bromine ratios are measured in neoplastic and normal kidney samples from humans by the particle induced X-ray emission analysis (PIXE) technique. It is found that cadmium which is normally present in the tubular cells of kidney is not detectable in tumor samples. It is also observed that the zinc-copper ratios in all neoplastic kidney tissues are decreased, but this observation cannot be extended to other element ratios.     

Effect of copper and zinc on the metabolism of N-nitrosamine and the activity of cytochrome P-450 in the liver of rats

The effect of copper and zinc on the metabolism of N-nitrosamine and activity of cytochrome P-450 in the liver of rats was studied. Copper and zinc enhanced obviously the activity of cytochrome P-450 in the liver. The level of cytochrome P-450 in the liver of control rats was 0.64 nmol/mg protein but that in the liver of rats treated with copper or zinc was 1.31 and 1.17 nmol/mg protein. There was a significant difference between the two groups (P less than 0.01-0.001). The activity of demethylase reflected the metabolic level of N-nitrosamine. In the control group, metabolic level of N-dimethyl-nitrosamine, N-methyl-N-butylacetonylnitrosamine and nitrosopyrrolidine was markedly higher than the copper and zinc groups. It is indicated that copper and zinc inhibit obviously the activity of demethylase, resulting in reduction of the metabolic activation of N-dimethylnitrosamine, N-methyl-N-butylacetonylnitrosamine and nitrosopyrrolidine. The role of copper and zinc in the chemical carcinogenesis is discussed.     

Microinjection of metallothionein-oncomodulin DNA into fertilized mouse embryos is correlated with fetal lethality

Oncomodulin (ONCO) is an oncodevelopmental protein expressed in placental and extraembryonic tissue and re-expressed in a wide variety of tumors. The metallothionein promoter (MT) is active in numerous adult tissues, in parietal and visceral extraembryonic endoderm, and developing liver. To study the function of oncomodulin we microinjected MT-ONCO DNA into one-cell embryos and examined tissues of fetal and adult mice. Analysis of implant sites from embryos, microinjected with MT-ONCO DNA then placed into pseudopregnant females, indicated a greater than three-fold increase in empty and necrotic implant sites relative to SV2NEO-microinjected embryos and a seven-fold rise relative to non-microinjected embryos. The striking feature of the lethality was the presence of a normal placenta but absence of fetal tissue. Few MT-ONCO DNA transgenic mice were isolated (3.5%) and none were able to express oncomodulin protein or RNA in any tissue examined, even after prolonged heavy metal stimulation of the MT promoter. Fetal mortality is best correlated with expression of oncomodulin causing an interruption of either cellular differentiation or organogenesis before day 9 in development.     

硒对慢性镉暴露致大鼠肾损伤的保护作用与机制

目的：探讨硒对慢性镉暴露致大鼠肾脏损伤毒性的保护作用及其机制。方法：雄性SD大鼠48只,根据体质量随机分为对照组、镉暴露组、硒对照组、硒干预组,每组12只,分别给予去离子水、氯化镉（3 mg/kg）、亚硒酸钠（0.01 mg/kg）、氯化镉（3 mg/kg）和亚硒酸钠（0.01 mg/kg）联合灌胃,每周灌胃6 d,共计12周。造模成功后,收集血液、尿液和肾脏组织,测定血清和尿液中尿素氮（UN）、肌酐（CREA）及尿蛋白含量等肾脏功能指标,观察肾脏组织病理结构改变,测定肾组织中活性氧（ROS）、丙二醛（MDA）、超氧化物歧化酶（SOD）及N-乙酰-β-D-葡萄糖苷酶（NAG）水平和活性;蛋白印迹法检测SETD6、DJ-1和Nrf2蛋白的表达。结果：与对照组比较,生化与病理检查结果显示镉暴露能导致明显的大鼠肾脏损伤,引起肾小球肿胀,肾小管广泛性病变,肾间质充血,炎性细胞浸润。肾组织ROS和MDA水平升高（P < 0.05）,总SOD活力下降（P < 0.05）,表明镉暴露诱导肾脏组织发生氧化应激损伤。而硒干预后减轻了镉暴露导致的肾脏组织结构和功能损伤,表明硒对镉暴露导致的肾脏损伤具有保护作用。同时,与对照组相比,SETD6、DJ-1和Nrf2在镉暴露后出现不同程度的蛋白表达下调（P < 0.05）;而硒干预后,与镉暴露组比较,SETD6蛋白表达显著下调（P < 0.05）,DJ-1和Nrf2蛋白表达显著上调（P < 0.05）,肾脏损伤减轻,表明硒可能通过抑制SETD6表达、增强DJ-1和Nrf2表达来发挥保护作用。结论：硒能有效拮抗慢性镉暴露导致的大鼠肾脏毒性,其作用机制可能是在氧化应激条件下,硒通过抑制SETD6,促进DJ-1表达,上调抗氧化转录因子Nrf2,增强机体抗氧化能力,减轻镉暴露诱导的氧化应激损伤。     

Tumor necrosis factor/cachectin decreases catalase activity of rat liver

Tumor bearing hosts and animals treated with endotoxin commonly show a decrease in the catalase activity of the liver and kidney. Since tumor necrosis factor (TNF)/cachectin may play a significant role in these conditions, we investigated its effects on the catalatic and peroxidatic activity of catalase in the liver and kidney of the rat. The activities of glucose-6-phosphate dehydrogenase and lactate dehydrogenase were measured simultaneously to monitor the pentose phosphate and glycolytic pathways, respectively. Injection i.p. of 100 micrograms/kg/day human recombinant TNF-alpha for 5 days resulted in a significant (P less than 0.01) decrease in the catalatic activity of the liver when compared to rats fed ad libitum. The decrease in four experiments ranged from 21 to 56%. A significant decrease (18%; P = 0.01) in liver catalatic and peroxidatic activity was also observed in another experiment using pair fed rats as controls. The peroxidatic activity of catalase with ethanol as hydrogen donor closely paralleled the catalatic activity. TNF treatment had no detectable effect on the catalatic or peroxidatic activity of catalase in the kidney. The activity of glucose-6-phosphate dehydrogenase increased (31-80%) significantly (P less than or equal to 0.02) in the liver and, to a lesser extent, in the kidney (5-27%, P = 0.05). Lactate dehydrogenase activity decreased (14-19%) significantly (P less than or equal to 0.05) in the liver and kidney but mainly in rats treated with TNF and additionally fasted for 24 h. Electron microscopic examination of liver sections showed that the hepatocytes of TNF-treated rats were undamaged but contained fewer and smaller peroxisomes than those of the control rats.     

Cisplatin-DNA adduct formation in maternal and fetal rat tissues after transplacental cisplatin exposure

Cis-diamminedichloroplatinum (II) (cisplatin), given to pregnantrats at 5 mg/kg body weight (bw) is a transplacental carcinogenfor fetal liver, kidney, nervous system and lung, resultingin tumor incidences of 22.5, 10.5, 6.1 and 7.5% respectively,in offspring grown to adulthood (B.A. Diwan et al., 1995, Toxicol.Appl. Pharm., 132, 115). In this study, the capacity of cisplatinto pass through the placental barrier and bind convalently toDNA in maternal and fetal tissues was evaluated. Pregnant F344/NOrats were injected i.p. with single does of 5, 10 or 15 mg cisplatin/kgbw at 18 days of gestation and sacrificed 24 h later. Cisplatin-DNAadducts were determined by dissociation-enhanced lanthanidefluroroimmunoassay (DELFIA) using both High (90 pmol/µgDNA) and low (0.50 pmol/µg DNA) Modified cisplatin-DNAstandards and atomic absorbance spectrometry (AAS). The adductquantities determined by the two DELFIAs varied in concert,but the DELFIA with Low Modified standard gave actual valuessimilar to those observed with AAS. In maternal and fetal tissues,with the exception of placenta in one experiment and maternalkidney in another experiment, the extent of cisplatin-DNA adductformation increased with dose. In maternal kidney, the low adductlevels observed at the 15 mg/kg dose may reflect kidney toxicity.Fetal kidney, liver and lung contained fewer cisplatin-DNA adductsthan the corresponding maternal tissues. In contrast, at 5 and15 mg/kg, fetal brain DNA contained higher adduct levels thanmaternal brain DNA. This study demonstrates the presence ofDNA damage induced by cisplatin in multiple maternal and fetalrat tissues at tumorigenic doses of drug; the results are thereforeconsistent with the hypothesis that genotoxic mechanisms playan important role in the drug-induced tumor incidence.     

Role of Atp7b gene in spontaneous and N-diethylnitrosamine-induced carcinogenesis in a new congenic strain, WKAH.C-Atp7b rats.

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b, we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEC rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEC rats. Incidences of spontaneous liver tumors in LEC, WKAH.C-Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEC rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C-Atp7b rats, and WKAH.C-Atp7b rats were more susceptible than WKAH rats. In an N-diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEC rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEC and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEC and WKAH.C-Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEC rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

Role of Atp7b Gene in Spontaneous and N-Diethylnitrosamine-induced Carcinogenesis in a New Congenic Strain, WKAH.C-Atp7b Rats

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b , we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEG rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEG rats. Incidences of spontaneous liver tumors in LEG, WKAH.C- Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEG rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C -Atp7b rats, and WKAH . C -Atp7b rats were more susceptible than WKAH rats. In an N -diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEG rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEG and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEG and WKAH.C- Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEG rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

Induction of hepatic metallothionein I in tumour-bearing mice.

Metallothionein (MT) is an intracellular metal-binding protein which has been implicated in various biological roles, including heavy-metal detoxification and zinc and copper homeostasis, and has putative antioxidant properties. High levels of MT have been detected in certain human tumours, but its functions are unclear. The presence of tumour may cause stress conditions along with alterations in host metabolism, such as the redistribution of metals and, subsequently, in changes in hepatic MT isoforms. The distribution of basal levels of MT-1 and MT-11 isoforms in livers of different strains of mice and their induction in mice inoculated with tumour cells are investigated. While Balb-c, C57/BL and CD1 mice strains had an equal distribution of both hepatic MT isoforms, MT-I and MT-II. In addition, MT-I was the predominant isoform synthesised (> 88%) in the livers of all strains of mice at 24 h after injection with either cadmium or zinc salts. After inoculation with human testicular T7800 or T7799 tumour cells, the major form of MT induced in the livers of nude (nu/nu) mice was Zn-MT-I, and its concentration was positively correlated with the size of the inoculated tumours (r2 = 0.85). A similar positive relation was found in the livers of Balb-c mice inoculated with MM45T mouse bladder tumour cells (r2 = 0.96). Following surgical removal of T7800 tumour, hepatic MT concentrations returned to basal values. There was an increase in plasma MT levels in tumour-bearing mice and it was positively correlated with the increase in hepatic MT levels. These results demonstrate a specific increase in hepatic MT-I isoform in tumour-bearing mice, and this may be due to a generalised stress during tumour growth.     

Formation of 8-hydroxydeoxyguanosine (8-OH-dG) in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA)

A significant increase of 8-hydroxydeoxyguanosine (8-OH-dG) was observed in the kidney DNA of rats given a renal carcinogen, the ferric complex of nitrilotriacetate (Fe-NTA) by single i.p. injection. By contrast, non- or weakly carcinogenic compounds, aluminum-nitrilotriacetate complex (Al-NTA), non-complexed NTA (Na2NTA) and ferric chloride had no effect on 8-OH-dG production in the kidney DNA. These results suggest the involvement of active oxygen radicals in Fe-NTA carcinogenesis.     

Dose-dependent enhancing effects of quinacrine on induction of preneoplastic glutathione S-transferase placental form positive liver cell foci in male F344 rats

Dose-dependent modifying effects of quinacrine on induction of preneoplastic liver cell foci were investigated in male F344 rats. Six week old animals were injected i.p. with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg, and starting 2 weeks later, rats were given quinacrine at dietary levels of 20, 100 and 500 p.p.m. for 6 weeks. Groups without either DEN or quinacrine treatment were used as controls. At week 3 following DEN administration, all animals were subjected to two-thirds partial hepatectomy, and after killing the animals at week 8, development of preneoplastic liver cell foci was investigated using the glutathione S-transferase placental form (GST-P) as a marker. The numbers and unit areas of GST-P-positive foci per cm2 were significantly increased in the DEN/quinacrine (500 p.p.m.) group as compared to DEN-alone group values. An increase in number was also evident in the 100 p.p.m. but not the 20 p.p.m. treated group, no lesions being induced by quinacrine alone (500 p.p.m.). Electron microscopic study confirmed that quinacrine dose-dependently induces lipidosis in hepatocytes, i.e. markedly myeloid lamellar cytoplasmic inclusion bodies were observed. The results thus demonstrated that quinacrine treatment enhances GST-P-positive liver cell foci development in a dose-dependent way, this effect presumably being related to the induction of lipidosis.     

Role of copper accumulation and metallothionein induction in spontaneous liver cancer development in LEC rats

The LEC rat spontaneously develops liver cancer after sufferingchronic liver injury caused by abnormal copper accumulationin the liver, but the role of copper accumulation in the inductionof liver, cancer remains obscure. We histochemically and biochemicallyexamined the content of copper and metallothionein (MT), a cytoplasmiccopper binding protein, in spontaneously developed preneoplasticand neoplastic liver lesions and compared them with those inthe surrounding liver tissues. Histochemically, the majorityof the preneoplastic liver lesions (68%) and liver cancers (59%)showed lower copper contents than the surrounding liver tissuesand no lesions were shown to accumulate more copper than thesurrounding tissues. A marked heterogeneity in copper stainingwas observed in cancer tissues. In contrast, these lesions showedan equal to higher MT content than their surroundings. Biochemicalmeasurements of copper and MT in cancer tissues supported thehistochemical findings. The bromodeoxyuridine (BrdU) labelingindex was high in all cancer tissues and some of the preneoplasticliver lesions. Parts of the cancer tissues with negative orweak staining for copper were highly labeled with BrdU. Takingthese results together, copper accumulation may exert a growthinhibitory effect on surrounding hepatocytes, whereas the hepatocytesin the liver lesions could proliferate, escaping from the effectof copper toxicity by increasing their MT induction and loweringcopper accumulation. Thus, accumulation of copper may act asa promoting factor for the development of liver cancer in LECrats by creating a selective growth environment.     

Differential expression and regulation of members of the cytochrome P450IA gene subfamily in human tissues

Cigarette smoking increases phenacetin O-deethylase (POD) activity in both the liver and placenta in man, but aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity is increased only in the placenta. Whilst there was no correlation between hepatic POD and AHH activities (rs = 0.42, P greater than 0.1), there was a highly significant correlation between these two activities in placenta (rs = 0.76, P less than 0.02). On Western blotting of human liver samples with an antibody specific to cytochrome P450IA2 in the rat, only the orthologue of P450IA2 could be detected. This antibody inhibited greater than 70% of hepatic high-affinity POD activity but had no effect on the placental activity. Furafylline, a methylxanthine that acts as a highly specific inhibitor of P450IA2-dependent activities in man, inhibited all of the high-affinity component of POD activity in human liver, but was at least three orders of magnitude less potent an inhibitor of placental POD and of both hepatic and placental AHH activities. As previously shown in the rat, exposure of man to polycyclic aromatic hydrocarbons, present in cigarette smoke, differentially induces P450IA2 in the liver and P450IA1 in extrahepatic tissues, at least in the placenta. Again, as in the rat, POD activity in the liver is catalysed by P450IA2, but in the placenta of women exposed to polycyclic aromatic hydrocarbons in cigarette smoke POD activity is catalysed by another isoenzyme, most likely P450IA1. Thus, tissue-dependent induction and substrate specificity of members of the P450IA family in man, at least in the placenta, appear to be the same as previously shown in the rat.