BackgroundTesticular germ cell tumors (TGCTs) respond well to cisplatin-based chemotherapy and show a low incidence of acquired resistance compared to most somatic tumors. The reasons for these specific characteristics are not known in detail but seem to be multifactorial. We have studied gene expression profiles of testicular and colon cancer derived cell lines treated with cisplatin. The main goal of this study was to identify novel gene expression profiles with their functional categories and the biochemical pathways that are associated with TGCT cells' response to cisplatin.ResultsGenes that were differentially expressed between the TGCT cell lines vs the (somatic) HCT116 cell line, after cisplatin treatment, were identified using the significance analysis of microarrays (SAM) method. The response of TGCT cells was strikingly different from that of HCT116, and we identified 1794 genes that were differentially expressed. Functional classification of these genes showed that they participate in a variety of different and widely distributed functional categories and biochemical pathways. Database mining showed significant association of genes (n = 41) induced by cisplatin in our study, and genes previously reported to by expressed in differentiated TGCT cells. We identified 37 p53-responsive genes that were altered after cisplatin exposure. We also identified 40 target genes for two microRNAs, hsa-mir-372 and 373 that may interfere with p53 signaling in TGCTs. The tumor suppressor genes NEO1 and LATS2, and the estrogen receptor gene ESR1, all have binding sites for p53 and hsa-mir-372/373. NEO1 and LATS2 were down-regulated in TGCT cells following cisplatin exposure, while ESR1 was up-regulated in TGCT cells. Cisplatin-induced genes associated with terminal growth arrest through senescence were identified, indicating associations which were not previously described for TGCT cells.ConclusionBy linking our gene expression data to publicly available databases and literature, we provide a global pattern of cisplatin induced cellular response that is specific for testicular cancer cell lines. We have identified cisplatin-responsive functional classes and pathways, such as the angiogenesis, Wnt, integrin, and cadherin signaling pathways. The identification of differentially expressed genes in this study may contribute to a better understanding of the unusual sensitivity of TGCT to some DNA-damaging agents.

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Hepatocytes isolated from Aroclor 1254 (PCB) pretreated rats metabolized 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to a reactive metabolite that induced DNA damage measured by alkaline elution or as increased unscheduled DNA synthesis. PhIP induced mutations in Salmonella typhimurium TA98 and DNA strand breaks and sister chromatid exchange(s) in Chinese hamster V79 cells co-incubated with PCB-hepatocytes. No, or only minor genotoxic, effects were observed when hepatocytes from non-induced rats were used. The bacterial mutagenicity could be inhibited by alpha-naphthoflavone, indicating a role of P-450 in the activation of PhIP. At least eight different metabolites could be separated on HPLC after PhIP had been incubated with PCB-hepatocytes. All of the directly acting mutagenicity towards S.typhimurium TA98 co-eluted with one of the metabolites. The identity of this metabolite was concluded to be 2-hydroxamino-PhIP based on the following evidence: (i) it reduced ferric ion to ferrous ion as hydroxylamines do, (ii) it had an identical UV spectrum and chromatographic properties as a species formed upon reduction of 2-nitro-PhIP by NADPH P-450 reductase. This product displayed a major peak at m/z 241 during thermospray mass spectrometry in the positive-ion mode as would be expected from 2-hydroxamino-PhIP. 2-Hydroxamino-PhIP was directly genotoxic both to TA98 and V79 cells. The genotoxic activity of the medium after removing the hepatocytes remained stable for several hours. Compared to 2-amino-3,4-dimethylimidazo[4,5-f]quinolone (MeIQ), PhIP caused a much larger increase in DNA damage in V79 cells (with hepatocyte activation), whereas MeIQ was more potent with respect to DNA damage induced in hepatocytes and bacteria.

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The nematocide and soil fumigant 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidney. It has been proposed that both cytochrome P-450 mediated activation and glutathione (GSH) conjugation pathways are operative in DNA damage and organotropy induced by DBCP. To determine the chemical mechanisms involved in the bioactivation of DBCP and to assess a role for an episulfonium ion intermediate, the mechanism of formation of GSH conjugate metabolites of DBCP was investigated. Five biliary GSH conjugates of DBCP were isolated from rats and identified by fast atom bombardment tandem mass spectrometry: S-(2,3-dihydroxy-propyl)glutathione (I), S-(2-hydroxypropyl)glutathione (IIA), S-(3-chloro-2-hydroxypropyl)glutathione (III), 1,3-di(S-glutathionyl)propan-2-ol (IV), and 1-(glycyl-S-cysteinyl)-3- (S-glutathionyl)propan-2-ol (V). The mechanisms of conjugate formation were addressed by assessing deuterium retention in conjugates derived from [1,1,2,3,3-2H5] DBCP (D5-DBCP). GSH conjugates I, III, IV, and V displayed quantitative retention of deuterium, an observation consistent with the formation of an episulfonium ion intermediate. GSH conjugate IIA, however, retained three atoms of deuterium, thus invoking a P-450 mechanism in its genesis. The involvement of glutathione transferase (GST) and sequential episulfonium ion intermediates in the formation of metabolites I, III, and IV was demonstrated in vitro. Upon incubation of DBCP with GST, metabolites I, III, and IV were identified by tandem mass spectrometry and were found to arise with quantitative retention of deuterium when D5-DBCP was employed as a substrate. An additional GSH conjugate, 1,2,3-tri(S-glutathionyl)propane (VI), was observed as the major metabolite in incubations of GST with DBCP. When the incubations of DBCP with GST were performed in H2(18)O, metabolite I incorporated two atoms of 18O, and metabolites III and IV incorporated one atom of 18O. The ability of GST to catalyze the formation of the four GSH conjugates observed in vivo, with quantitative retention of deuterium and incorporation of 18O from H2(18)O, may be rationalized by a mechanism invoking the initial formation of S-(2-bromo-3-chloropropyl)glutathione. Rearrangement of this unstable conjugate via several reactive episulfonium ions, with either hydrolysis by water or alkylation of GSH at various stages, would account for the pattern of metabolites and their status of isotopic enrichment observed under various incubation conditions.(ABSTRACT TRUNCATED AT 400 WORDS)