Assaying potential carcinogens with Drosophila.
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Drosophila offers many advantages for the detection of mutagenic activity of carcinogenic agents. It provides the quickest assay system for detecting mutations in animals today. Its generation time is short, and Drosophila is cheap and easy to breed in large numbers. The simple genetic testing methods give unequivocal answers about the whole spectrum of relevant genetic damage. A comparison of the detection capacity of assays sampling different kinds of genetic damage revealed that various substances are highly effective in inducing mutations but do not produce chromosome breakage effects at all, or only at much higher concentrations than those required for mutation induction. Of the different assay systems available, the classical sex-linked recessive lethal test deserves priority, in view of its superior capacity to detect mutagens. Of practical importance is also its high sensitivity, because a large number of loci in one fifth of the genome is tested for newly induced forward mutations, including small deletions. The recent findings that Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver makes the organism eminently suitable for verifying results obtained in prescreening with fast microbial assay systems. An additional advantage in this respect is the capacity of Drosophila for detecting short-lived activation products, because intracellular metabolic activation appears to occur within the spermatids and spermatocytes. (+info)
The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice.
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Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis. (+info)
Prokaryotic expression and characterization of human AP DNA endonuclease.
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The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 microM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties. (+info)
Beta-catenin mutations are frequent in hepatocellular carcinomas but absent in adenomas induced by diethylnitrosamine in B6C3F1 mice.
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Activating mutations in the region of the beta-catenin gene corresponding to the NH2-terminal phosphorylation sites of glycogen synthetase kinase 3beta have been causally implicated in carcinogenesis. In this study, the beta-catenin exon 3 was examined in hepatic lesions induced by diethylnitrosamine in B6C3F1 mice. PCR and DNA sequencing detected seven beta-catenin mutations in 13 samples dissected from hepatocellular carcinoma tissues, but none in 14 hepatic adenomas. All of the mutations were found in codon 41 encoding a threonine residue, one of the possible glycogen synthetase kinase-3beta phosphorylation sites. Although beta-catenin protein was immunohistochemically stained mainly on the cell membrane in preneoplastic hepatocytic foci and most adenomas, as observed in normal hepatocytes, it was detected in the cytoplasm and nuclei in addition to the cell membrane, indicating stabilization of the protein in HCCs. This shift in staining was observed not only in tumors with mutations, but also in examples lacking exon 3 mutations. Our data demonstrate that beta-catenin alterations may be important for malignant progression during multistep hepatic carcinogenesis in mice. (+info)
Nordihydroguairetic acid is a potent inhibitor of ferric-nitrilotriacetate-mediated hepatic and renal toxicity, and renal tumour promotion, in mice.
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Ferric-nitrilotriacetate (Fe-NTA) is a known renal carcinogen. In the present study, we report the effect of a potent lignin-derived herbal antioxidant, nordihydroguairetic acid (NDGA), against Fe-NTA-mediated tissue toxicity. Fe-NTA (alone) treatment of mice enhances ornithine decarboxylase activity to 259% in liver and 341% in kidney and increases [3H]thymidine incorporation in DNA to 250% in liver and 324% in kidney compared with the corresponding saline-treated controls. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 138 and 123%, respectively, in liver at a higher dose of 2 mg NDGA/day/animal whereas in kidney the reduction was to 118 and 102%, respectively, compared with the corresponding saline-treated controls. In the Fe-NTA (alone)-treated group, a 12% renal tumour incidence was recorded whereas, in N-diethylnitrosamine (DEN)-initiated and Fe-NTA-promoted animals, the percentage tumour incidence was increased to 68% as compared with untreated controls. No tumour incidence was recorded in the DEN-initiated, non-promoted group. The administration of NDGA, afforded >80% protection against DEN- and Fe-NTA-mediated renal tissue injury in vivo. Fe-NTA treatment also enhanced hepatic and renal microsomal lipid peroxidation to 170 and 205% of saline-treated controls, respectively, and hydrogen peroxide generation by >2.5-fold in both tissues accompanied by a 51 and 21% decrease in the level of glutathione and 35-48 and 35-50% decrease in the activities of glutathione-metabolizing and antioxidant enzymes in liver and kidney, respectively. These changes were reversed significantly in animals receiving a pre-treatment of NDGA. Our data show that NDGA can abrogate the toxic and tumour-promoting effects of Fe-NTA in liver and kidney of mice and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and tumorigenesis. (+info)
Correlation of changes in natural killer cell activity and glutathione S-transferase placental form positive hepatocytes in diethylnitrosamine-induced rat hepatocarcinogenesis.
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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. (+info)
Progression of hepatic neoplasia in medaka (Oryzias latipes) exposed to diethylnitrosamine.
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Progression of hepatic neoplasia was assessed in medaka (Oryzias latipes) following aqueous exposure to diethylnitrosamine (DEN). Larvae (2 weeks old) were exposed to 350 or 500 p.p.m. DEN for 48 h, while adults (3-6 months old) were exposed to 50 p.p.m. DEN for 5 weeks. Fish were maintained as long as possible to determine malignant potential of resultant neoplasms. A total of 423 medaka with 106 hepatic neoplasms were examined. There were marked differences in tumor prevalence between exposure groups including: (i) higher prevalence of hepatocellular carcinomas in medaka exposed as adults (100% of hepatocellular tumors in adult-exposed medaka were malignant, while only 51.5% of larval hepatocellular tumors were malignant); (ii) higher prevalence of biliary tumors in medaka exposed as larvae (46.4% of all tumors in larval-exposed medaka were biliary versus 8.1% in adult-exposed fish); (iii) higher prevalence of mixed hepato-biliary carcinomas in adult-exposed medaka (24.3%) compared with those exposed as larvae (3%). In addition, a unique hepatocellular lesion termed 'nodular proliferation' was only observed in adult-exposed medaka. The lesion was characterized by small size (50-300 microm), complete loss of normal tubular architecture and variable megalocytosis. Nodular proliferation was distinct from preneoplastic foci of cellular alteration and may represent microcarcinomas. There was a step-wise increase in mean diameter with age (days post-exposure) from nodular proliferation (174 microm, 17 days) to hepatocellular carcinoma (1856 microm, 62 days) and mixed carcinomas (3209 microm, 93 days) in adult-exposed medaka. Metastasis was observed with 19 neoplasms and tumors with the highest metastatic potential were hepatocellular and mixed carcinomas. The most common form of metastasis was trans-coelomic, followed by direct invasion and distant metastasis, presumably via the vascular route. Differences in tumor prevalence between exposure groups were believed to be the result of length of DEN exposure rather than age of fish at the time of exposure. In larval medaka with brief (48 h) DEN exposure, neoplasms are thought to be the result of dedifferentiation of hepatic cells, with slow progression of foci of cellular alteration to benign and then malignant tumors. In contrast, with adult medaka and prolonged (5 week) DEN exposure, neoplasms are believed to result from initiation of committed stem cells and formation of microcarcinomas ('nodular proliferation'), before progressing to larger hepatocellular and then mixed carcinomas. (+info)
The effect of 1/3 partial hepatectomy on the growth of glutathione S-transferase positive foci.
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Our previous studies indicated that glutathione S-transferase 7-7 (GST 7-7) positive foci induced after initiation have a lower threshold towards proliferative stimuli compared with surrounding hepatocytes. This observation would predict that persistent growth stimuli of low intensity could be very effective in promoting the emergence of focal lesions. To test this possibility, the present study was designed to determine the effect of 1/3 partial hepatectomy (PH) on the incidence and growth of foci in initiated rat liver. The rationale for using a 1/3 PH was that it is known to induce a proliferative response which is less intense but more prolonged compared with that elicited by 2/3 PH. Male Fischer 344 rats (110-120 g) were initiated with diethylnitrosamine (200 mg/kg, i.p.). Three weeks later 1/3 PH (median lobe), 2/3 PH (median and left lobes) or sham operation (SH) was performed. An additional group of initiated animals had the median lobe and the left lobe of the liver removed sequentially (1/3 + 1/3 PH), 3 weeks apart. All rats were killed 8 weeks after carcinogen administration. The results indicated that the number of GST 7-7 positive foci was similar in all groups; however, the percent area occupied by foci was increased in rats receiving 2/3 PH compared with SH (0.21 +/- 0. 08 versus 0.09 +/- 0.03). Interestingly, 1/3 PH was nearly as effective as 2/3 PH in stimulating the growth of foci (percent area 0.18 +/- 0.06 versus 0.21 +/- 0.08), although the magnitude of the stimulus is only half for the former group compared with the latter; peak labeling index was 19 +/- 6 with 1/3 PH compared with 40 +/- 2 with 2/3 PH. Moreover, the maximum increase in the size of foci (percent area 0.37 +/- 0.12) was achieved when the median and left lobes were removed sequentially, three weeks apart. These results indicate that persistent growth stimuli of low intensity can be very effective in promoting the growth of focal lesions. (+info)