Age-dependent carcinogenic susceptibility in rat liver is related to potential of gap junctional intercellular communication

Connexin 32 (Cx32) is a major gap junction protein in the liver. The authors previously demonstrated that transgenic rats carrying a dominant negative mutant of Cx32 (Cx32ΔTg) have much decreased capacity for gap junctional intercellular communication (GJIC) and increased susceptibility to diethylnitrosamine (DEN)-induced hepatocarcinogenesis as compared to littermate wild-type (wt) rats. To evaluate the age-dependent susceptibility to DEN-induced hepatocarcinogenesis and alteration of GJIC function, male Cx32ΔTg and wt rats at 10, 30, or 85 weeks old were given a single intraperitoneal administration of DEN (40 mg/rat) and sacrificed 12 weeks later. The number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic foci were significantly increased in the liver of 10- and 30-wk-old Cx32ΔTg rats compared with age-matched wt. However, in the 85-wk-old rats, both Cx32ΔTg and wt rats had similarly large number and area of GST-P-positive foci, and the difference was not significant. Interestingly, function of hepatic GJIC was reduced and protein and mRNA expression of Cx32 were decreased with aging in wt rats. These results suggest that a decline of hepatic intercellular communication through gap junction results in increased susceptibility to DEN-induced hepatocarcinogenesis in aged rats.     

Tomato oleoresin inhibits DNA damage but not diethylnitrosamine-induced rat hepatocarcinogenesis

Various studies have shown that lycopene, a non-provitamin A carotenoid, exerts antioxidant, antimutagenic and anticarcinogenic activities in different in vitro and in vivo systems. However, the results concerning its chemopreventive potential on rat hepatocarcinogenesis are ambiguous. The aim of the present study was to investigate the antigenotoxic and anticarcinogenic effects of dietary tomato oleoresin adjusted to lycopene concentration at 30, 100 or 300 ppm (administered 2 weeks before and during or 8 weeks after carcinogen exposure) on liver of male Wistar rats treated with a single intraperitoneal dose of 20 or 100mg/kg of diethylnitrosamine (DEN), respectively. The level of DNA damage in liver cells and the development of putative preneoplastic single hepatocytes, minifoci and foci of altered hepatocytes (FHA) positive for glutathione S-transferase (GST-P) were used as endpoints. Significant reduction of DNA damage was detected when the highest lycopene concentration was administered before and during the DEN exposure (20mg/kg). However, the results also showed that lycopene consumption did not reduce cell proliferation in normal hepatocytes or the growth of initiated hepatocytes into minifoci positive for GST-P during early regenerative response after 70% partial hepatectomy, or the number and area of GST-P positive FHA induced by DEN (100mg/kg) at the end of week 10. Taken together, the data suggest a chemopreventive effect of tomato oleoresin against DNA damage induced by DEN but no clear effectiveness in initiating or promoting phases of rat hepatocarcinogenesis.     

Correlation of changes in natural killer cell activity and glutathione S-transferase placental form positive hepatocytes in diethylnitrosamine-induced rat hepatocarcinogenesis.

To evaluate the induction of preneoplastic hepatic foci in relation to natural killer cell (NK) activity, we sequentially analyzed glutathione S-transferase placental form positive (GST-P+) hepatocytes and NK activity during diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis in Sprague-Dawley rats. Previous studies have shown that NK activity can modulate the carcinogenic process induced by chemical carcinogens. Newborn females were initially given a single intraperitoneal injection of 15 mg DEN/kg and three weeks later, they were treated with 500 ppm phenobarbital (PB). From week 3, PB was administered in drinking water for 9 weeks. Interim and terminal sacrifices were performed at weeks 12, 15 and 30. GST-P+ hepatocytes increased with age in DEN-treated rats, especially in the population of more than two GST-P+ hepatocytes. The NK activity of DEN-treated rats did not significantly differ from that of control rats until week 12, but it progressively decreased from week 15 to 30. These results indicate that changes of NK activity inversely correlated with the induction of preneoplastic hepatic foci. This strong correlation of decreased NK activity with enhanced induction of GST-P+ foci suggests that NK activity is important in the early progression of hepatocarcinogenesis in rats.     

Quantitation of the cancer process in C57BL/6J, B6C3F1 and C3H/HeJ mice

This study examines growth alterations in liver foci and tumor development as a basis for the different susceptibility in hepatocarcinogenesis found among different strains of mice. Male C57, B6C3F1, and C3H mice treated with a single dose (1 mg/kg) of N,N-diethylnitrosamine (DEN) at 15 days of age and followed up to 12 months displayed a strain-dependent (C3H > B6C3F1 > C57) increase in incidence, number, volume fraction, and size of foci and macroscopic lesions (masses). DEN-treated mice exhibited a time-dependent increase in foci size but not in foci number. Phenobarbital (PB) treatment (500 ppm) in the drinking water starting 2 weeks after DEN-initiation did not affect the incidence or number of masses and foci. In all 3 strains, the bromodeoxyuridine labeling index in foci correlated with foci growth, supporting the major role of cell proliferation in foci growth. Measurements of apoptosis by morphological criteria with H&E staining suggest that intrafocal apoptosis may be a late event preventing foci growth and possibly also promoting focal cell selection, whereas extrafocal apoptosis may facilitate clonal growth by removing adjacent normal cells. The onset of conversion of foci to masses also correlated with strain susceptibility to hepatocarcinogenesis.     

Transgenic mice expressing hepatitis B virus X protein are more susceptible to carcinogen induced hepatocarcinogenesis

The hepatitis B virus X (HBx) protein is thought to be implicated in the development of human hepatocellular carcinoma (HCC), but its exact function remains controversial. To investigate whether the expression of the HBx gene alone can induce HCC on an inbred C57BL/6 strain that displays a lower spontaneous rate of liver cancer, and to determine if HBx transgenic mice are more susceptible to the effects of hepatocarcinogens, C57-TgN (HBx) X transgenic mice were bred with normal C57BL/6 mice strain. The F1 mice (about 50% HBx positive and 50% HBx negative) were treated with a single dose of diethylnitrosamine (DEN) at 7 days of age, or were untreated. Mice were killed at appropriate time points and were analyzed for histological change in the liver. The expression of HBx protein were examined by using immunohistochemical staining. Glycogen storage foci were examined by using periodic acid-Schiff (PAS) staining. In HBx transgenic mice untreated with DEN, HBx expression and glycogen storage foci were always observed in the liver after 8 weeks, but not obvious histological pathologic changes. Histological examination of liver tissue confirmed that DEN-treated HBx mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Hepatocellular adenomas and carcinomas were also more frequent in DEN-treated HBx-positive than HBx-negative mice. Taken together, our results suggest that HBx gene expression alone is not sufficient for carcinogenesis, but may act as a promoter for malignant transformation.     

Articular chondromatosis and chrondroid metaplasia in transgenic TAg mice

The C3(1)/SV40 T antigen transgenic mouse model for which rapid mammary and prostate tumor development has been documented uses the FVB/N mouse as a background strain. In this study, where the background strain used was the C57BL/6J mouse, neither mammary nor prostate tumors developed over periods of up to 40 weeks. However, a disturbance of hyaline cartilage in joints was observed similar to that found in synovial chondromatosis in humans. In addition, cartilage thickening in the external ears and cartilaginous metaplasia of the ascending aorta also occurred. This suggests that rearrangement of the transgene occurred in breeding on the C57BL background, thus modifying its expression. It raises the possibility that the genetic changes induced by the SV40 T antigen transforming sequence are important in cartilage homeostasis.     

Possible prevention by abieslactone of development of diethylnitrosamine-initiated GST-P positive foci in the rat liver

Triterpenoid compounds, isolated from plants of Abies genus (Pinceae), are known to exert anti-tumor promotion activities in mouse skin carcinogenesis. In the present study, we investigated whether AVB-1 and acid and acid methyl ester derivatives have inhibitory effects on rat hepatocarcinogenesis by using a liver medium-term bioassay for carcinogens (Ito's test), immunohistochemically assessing the numbers and areas per cm(2) of preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci. In experiment 1, 6-week-old male Fisher 344 rats were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) and subjected to two-thirds partial hepatectomy at week 3. From weeks 2 to 8, the compounds were given three times a week at a dose of 1 mg/kg b.w. by i.g. gavage and found to significantly decrease the number of GST-P-positive foci in the liver. In experiment 2, AVB-1 was given three times a week at doses of 3, 1, or 0.3 mg/kg b.w. by i.g. gavage from weeks 2 to 8. All doses of AVB-1 significantly decreased the numbers of GST-P-positive foci. Thus, our results suggest that AVB-1 is a chemopreventive agent for rat hepatocarcinogenesis.     

Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or N-nitrosobis(2-hydroxypropyl)amine by a choline-deficient, L-amino acid-defined diet administered prior to the carcinogen exposure in rats.

Effects of pre-administration of a choline-deficient, L-amino acid-defined (CDAA) diet on hepatocarcinogenesis initiated with diethylnitrosamine (DEN) or N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated. A pre-administrating period was set as 1 week, because CDAA diet induces liver injuries by this time-point. In a time-course study, male Fischer 344 rats, 6 weeks old, received a 1-week pre-administration of choline-supplemented, L-amino acid-defined (CSAA) or CDAA diet, DEN at a dose of 100 mg/kg body weight by a single intraperitoneal injection, then CSAA or CDAA diet for up to 8 weeks, and were sacrificed 4, 6 and 8 weeks after DEN. CDAA diet administered only after DEN significantly increased the numbers of glutathione S-transferase placental form (GST-P)-positive lesions 4, 6 and 8 weeks after DEN and their sizes 6 and 8 weeks after DEN. CDAA diet administered both before and after DEN similarly increased the numbers and sizes of GST-P-positive lesions, but with a significantly greater degree than obtained by the diet administered only after DEN. In a dose response study, rats received vechicle or DEN, at a dose of 0.001, 0.01, 0.1, 1, 10, 20, 50, 100 or 200 mg/kg body weight, 1 week after the commencement of CSAA or CDAA diet, and sacrificed 8 weeks after vehicle or DEN. The significant increases of the numbers of GST-P-positive lesions were obtained after 50-200 mg/kg body weight of DEN under the CSAA diet administration, whereas those were detected after 10-200 mg/kg under CDAA diet administration. Sizes became significantly larger with only 200 mg/kg body weight of DEN in the CSAA case but with 50-200 mg/kg in the CDAA case. Male Wistar rats received a 1-week pre-administration of CSAA or CDAA diet, vehicle or BHP, at a dose of 600 or 1200 mg/kg body weight, by a single intraperitoneal injection, then CSAA or CDAA diet for 8 weeks, and were then sacrificed. The numbers of GST-P-positive lesions demonstrated significant increment with 1200 mg/kg body weight of BHP by CDAA diet administered only after BHP and, to a significantly greater degree, by the diet administered both before and after BHP. While CDAA diet administered only after BHP did not alter the sizes of GST-P-positive lesions, the diet administered both before and after 600 and 1200 mg/kg body weight of BHP significantly increased the sizes of the lesions. These results indicate that the pre- plus post-administration of CDAA diet enhances hepatocarcinogenesis initiated with DEN or BHP, more than the post-administration only, thus providing a sensitive model to detect weak liver carcinogenic potency of environmental chemicals.     

Strain background alters mammary gland lesion phenotype in transforming growth factor-alpha transgenic mice

Whey acidic protein (WAP)-transforming growth factor (TGF)-alpha transgenic mice acquire both cancerous and noncancerous mammary lesions. For this study, we evaluated the effect of mouse strain background on the incidence, latency, and histotype of two noncancerous lesions, hyperplastic alveolar nodules (analogous to typical hyperplasias in women), and macrocysts. These lesions display characteristics of fibrocystic changes observed in breasts of women, and in both mice and humans are associated with an uncertain risk of progression to neoplasia. Virgin transgenic mice of the (C57BL/6J;SJL)F2 background developed very few hyperplastic alveolar nodules and no macrocysts. In contrast, when the WAP-TGF-alpha transgene was carried on the FVB/N strain, congenic virgin transgenic mice acquired both lesion types with approximately 100% penetrance. In the (FVB;C57BL/6J)F1 background, hyperplastic alveolar nodule incidence was reduced to approximately the nontransgenic mouse level, and macrocyst latency was increased dramatically. Crossing into C57BL/6 resulted in elimination of the macrocyst phenotype. Finally, FVB strain transgenic mammary epithelium transplanted into nontransgenic recipients of the FVB/N or (FVB;C57BL/6J)F1 backgrounds displayed macrocyst latency characteristic of the recipient, and not donor, mouse strain. Quantitative real-time polymerase chain reaction analysis demonstrated that, despite the difference in macrocyst incidence between (FVB;C57BL/6J)F1 and C57BL/6 virgin transgenic mice (81% versus 0%), the level of TGF-alpha expression was not different. FVB strain transgenic mice expressed only twofold more TGF-alpha than the other backgrounds. These findings indicate that C57BL/6J modifier alleles inhibit mammary lesion incidence and macrocyst latency in a semidominant manner, and that suppression of lesion development can involve host factors that are independent of mammary epithelial genotype.     

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.     

Difference in glucose intolerance between C57BL/6J and ICR strain mice with streptozotocin/nicotinamide-induced diabetes

Blood glucose and plasma insulin levels between C57BL/6J and ICR strain mice with nicotinamide (NA) and streptozotocin (STZ)-induced diabetes were compared to establish a suitable strain of the experimental diabetic mouse model. The mice were intraperitoneally treated twice with STZ (100 mg/kg) 15 min after injection of NA (120 mg/kg) at a 1-day interval, and non-fasting blood glucose level was then weekly monitored for 5 weeks. The blood glucose level in ICR mice gradually increased and was about 2-times higher than that in C57BL/6J mice at the end of the observation. The plasma insulin level in ICR mice was comparatively low, compared with that in C57BL/6J mice. ICR mice were also markedly glucose-intolerant when oral glucose tolerance test was performed. These results indicate that ICR strain is more sensitive than C57BL/6J strain as a mouse model with NA/STZ-induced mild diabetes.     

Lack of modification of rat hepatocarcinogenesis by fernane-type triterpenoids,isolated from a Euphorbia genus

Inhibitors of topoisomerases, enzymes that produce an unusual type of DNA damage, are considered as antitumor agents. Recently it has been reported that the fernane-type triterpenoid EC-2 and its hydroxyl derivative, isolated from Euphorbia, are potent topoisomerase II inhibitors. In this study, the modifying effects of EC-2 and EC-4 on the development of putative preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci, in the liver of rats were investigated using a medium-term bioassay system. Fisher 344 male, 6-week-old rats were given a single intraperitoneal injection (200 mg/kg b.w.) of diethylnitrosamine or saline at the beginning of the experiment and subjected to 2/3 partial hepatectomy at the 3rd week. The test compounds were administered five times/week by i.g. gavage at a dose of 1 mg/kg b.w. from 2 to 8 weeks. Quantitation of the numbers and areas per cm(2) of induced GST-P positive foci did not demonstrated any significant differences among the groups and no variation in cell proliferation as indicated by 5-bromo- 2'-deoxyuridine (BrdU) labeling. Our results suggest that EC-2 and EC-4 have no modifying effects on rat hepatocarcinogenesis.     

Effects of fasting and intermittent fasting on rat hepatocarcinogenesis induced by diethylnitrosamine

The influences of fasting on DEN-initiation and of intermittent fasting (IF) on the rat liver chemical carcinogenesis process were evaluated in a 52-week long assay. Three groups of adult male Wistar rats were used: Groups 1 to 3 were treated with a single i.p. injection of 200 mg/kg of diethylnitrosamine (DEN). Group 2 was submitted to 48 h fasting prior to DEN treatment. After the 4th week, Group 3 was submitted to IF, established as 48 h weekly fasting during 48 weeks, while Groups 1 and 2 were fed ad libitum until the 52nd week. All animals were submitted to 70% partial hepatectomy and sacrificed at the 3rd and 52nd weeks, respectively. Fasting prior to DEN-initiation did not influence the development of altered foci of hepatocytes (AFHs) and of hepatic nodules (Group 2 vs. Group G1). IF inhibited the development of preneoplastic lesions, since this dietary regimen decreased the number and the size of glutathione S-transferase (GST-P) positive foci and the number and size of liver nodules (Group G3 vs. Group G1). The inhibitory effect of IF was also reflected in the development of clear and basophilic cell foci. These results indicate that long-term IF regimen exerts an anti-promoting effect on rat hepatocarcinogenesis induced by DEN.     

化疗治愈荷瘤小鼠模型的建立及氟尿嘧啶抑瘤作用的免疫机制

目的:建立荷瘤野生型C57BL/6小鼠及C57BL/6裸鼠模型,探讨5-氟尿嘧啶(5-FU)抑瘤作用的免疫机制。方法:给正常野生型C57BL/6小鼠皮下接种小鼠淋巴瘤EL4细胞,建立荷EL4肿瘤的小鼠模型。接种瘤细胞后12d,荷瘤小鼠腹腔单次注射不同剂量的5-FU,找出5-FU可发挥最大的抑瘤作用、并能致使荷瘤小鼠肿瘤消退不再复发的最小使用剂量。遗传背景相同的野生型C57BL/6小鼠及C57BL/6裸鼠同时皮下接种小鼠淋巴瘤EL4细胞,建立两种荷瘤小鼠模型。接种瘤细胞后12d,两种荷瘤小鼠腹腔内同时注射可使野生型C57BL/6荷瘤小鼠肿瘤消退不再复发5-FU的最低剂量,以正常野生型C57BL/6小鼠为对照,观察5-FU对T淋巴细胞缺陷裸鼠的抑瘤作用。结果:以75mg/kg5-FU治疗1周后,两种荷瘤小鼠的肿瘤以相同的速率缩小直至消失。但在5-FU治疗2周后,T淋巴细胞缺陷的C57BL/6裸鼠肿瘤复发,而免疫功能正常的野生型C57BL/6小鼠肿瘤治愈。结论:单一剂量的5-FU对荷淋巴瘤EL4小鼠具有明显的近期治疗效果,5-FU抑瘤作用的发挥不需要T细胞参与;但5-FU抗肿瘤作用的远期疗效与机体T淋巴细胞功能密切相关。     

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.     

Analysis of carcinogenic activity of some pesticides in a medium-term liver bioassay in the rat.

Eight pesticides were tested in a medium-term bioassay based upon the induction of preneoplastic lesions in the liver. Rats were initially given diethylnitrosamine intraperitoneally at a dose of 200 mg/kg body weight and 2 weeks later were treated with the pesticides for 6 weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione S-transferase placental form positive foci in the liver with those of controls given diethylnitrosamine (DEN) alone. Positive results were seen with p,p-DDT and Triadimefon. Permethrin (mixture of 39% cis form and 61% trans form) showed borderline results. Permethrin (25/75), Deltamethrin, Cypermethrin (52/48), while Trimorphamide and Propineb gave negative results. Our findings provide experimental evidence to indicate that compounds active in this assay have a potential for liver carcinogenicity in rodents.     

Pathology of diethylnitrosamine toxicity in the fish Rivulus marmoratus

Rivulus marmoratus were exposed to 0, 10, 21, 45, 95, or 200 mg/liter diethylnitrosamine (DEN) for 6 weeks and examined 12 weeks after the end of exposure. Fatty change, hepatocellular glycogenosis, multiple basophilic foci, enlarged and distorted cells with or without an enlarged nucleus, and hyaline bodies and cytological alterations observed after exposure to DEN. Hemangiomas, cholangiomas, biliary cystadenomas, and glandular, trabecular and anaplastic hepatocellular carcinomas were observed at the 18th week. Only those fish exposed to 95 mg/liter DEN had cavernous hemangiomas and peliosis-like lesions, which could be a preneoplastic lesion preceding cavernous hemangiomas. Adenomatous hyperplasia of thyroid and granulomas were other chronic reactions caused by DEN toxicosis.     

Amphetamine-induced toxicity in dopamine terminals in CD-1 and C57BL/6J mice: complex roles for oxygen-based species and temperature regulation.

In order to examine differential strain susceptibility to neurotoxic effects of amphetamine and to assess the potential role of superoxide radicals in amphetamine-induced dopaminergic damage, the drug was injected to mice with different levels of copper/zinc superoxide dismutase (Cu/Zn SOD) enzyme. Administration of amphetamine (10 mg/kg, i.p., given every 2 h, a total of four times) to wild-type CD-1 and C57BL/6J mice caused significant decreases in dopamine and 3,4-dihydroxyphenylacetic acid levels, in [(125)I]RTI-121-labeled dopamine transporters as well as a significant depletion in the concentration of dopamine transporter and vesicular monoamine transporter 2 proteins. The amphetamine-induced toxic effects were less prominent in CD-1 mice, which have much higher levels of Cu/Zn SOD activity (0.69 units/mg of protein) in their striata than C57BL/6J animals (0.007 units/mg of protein). Transgenic mice on CD-1 and C57BL/6J background, which had striatal levels of Cu/Zn SOD 2.57 and 1.67 units/mg of protein, respectively, showed significant protection against all the toxic effects of amphetamine. The attenuation of toxicity observed in transgenic mice was not caused by differences in amphetamine accumulation in wild-type and mutant animals. However, CD-1-SOD transgenic mice showed marked hypothermia to amphetamine whereas C57-SOD transgenic mice did not show a consistent thermic response to the drug.The data obtained demonstrate distinctions in the neurotoxic profile of amphetamine in CD-1 and C57BL/6J mice, which show some differences in Cu/Zn SOD activity and in their thermic responses to amphetamine administration. Thus, these observations provide evidence for possible complex interactions between thermoregulation and free radical load in the long-term neurotoxic effects of this illicit drug of abuse.     

Downregulation of apoptosis revealed by laser microdissection and cDNA microarray analysis of related genes in rat liver preneoplastic lesions

Regulation of cell proliferation and apoptosis plays a key role in tumor development. To elucidate mechanisms underlying hepatocarcinogenesis, patterns of gene expression, including apoptosis-related genes, were compared between glutathione S-transferase placental form (GST-P)-positive preneoplastic foci and surrounding tissue in the rat. Lesions were induced with a single intraperitoneal injection of diethylnitrosamine (DEN, 200mg/kgbw) and then 100ppm 2-acetylaminofluorene (2-AAF)-containing diet combined with two-thirds partial hepatectomy. Frozen sections of the livers were applied for immunohistochemical staining of GST-P, and both positive foci and surrounding negative areas were harvested by laser microdissection. Total RNAs were extracted and amplified with T7 polymerase to allow gene expression analysis by cDNA microarray assays. In the GST-P-positive foci, altered levels were observed for many genes, mostly related to metabolism or catalysis, with increased expression of testosterone-repressive prostate message-2 (TRPM-2), which is reported to act as a protective factor against apoptosis, and decreased expression of thymus-expressed acidic protein (TEAP), which is considered to promote apoptosis. The results indicate that rat liver preneoplastic lesions might be protected against apoptosis and that the approach adopted is useful for clarification of mechanisms underlying hepatocarcinogenesis.