Molecular toxicology最新文献

The present study was undertaken to isolate and identify various lipids bound to 14C label during hepatic microsomal metabolism of 14CCl4 in vitro under anaerobic conditions and in vivo in rats. The two major radioactive fractions identified by thin-layer chromatography each for neutral lipids and phospholipids from in vitro and in vivo experiments corresponded to fatty acids and triglycerides and to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Approximately 89% of the radioactivity associated with phospholipids was found in PC and PE fractions. Hydrolysis of PC and PE with phospholipase A2 (EC 3.1.1.4) released about 50% of the total radioactivity as lipid moieties corresponding to fatty acids. The radioactive neutral lipids and the lipid moieties hydrolyzed from PC and PE were methylated with boron trifluoride in methanol. These methylated lipids were separated by reversed-phase high-performance liquid chromatography (HPLC), and the elution profiles of 14C label found for the lipids obtained from in vitro experiments were similar to those from in vivo. The major radioactive fractions eluted immediately after methyl oleate were identified as trichloromethyloctadecenoic and trichloromethyleicosatrienoic acid methyl esters by chemical ionization mass spectrometry. The mass spectral analysis of these fractions also indicated the formation of dichlorocarbene adduct of oleic acid. However, similar mass spectrometric detection of trichloromethylated lipids was not evident in neutral lipids and phospholipids isolated from in vivo studies. The 14C-labeled lipids eluted as a nonpolar fraction exhibited a high molecular weight containing more than three chlorines. Dimerization and cross-linking of trichloromethylated lipids based on HPLC and mass spectral analysis are also discussed in this paper.

Four new peaks were observed upon amino acid analysis of alpha 1-proteinase inhibitor (alpha 1-PI), which was inactivated by acrolein under in vitro conditions. The first peak emerged just before ammonia, the second and third between ammonia and lysine, and the fourth between histidine and arginine. The new fourth peak was also observed when model compounds of lysine (N-acetyllysine or polylysine) were reacted with acrolein and subsequently processed for amino acid analysis. This new compound was purified by high-voltage paper electrophoresis and subjected to fast atom bombardment mass spectrometry, which showed a protonated molecule ion at m/z 203 [M + H]+ followed by m/z 186 [M + H+ - NH3]+. This compound was thus identified as 3-oxopropyllysine, a lysine adduct of acrolein. Similarly, when a model polypeptide of histidine, polyhistidine, was reacted with acrolein under the same conditions as alpha 1-PI, three new peaks (besides histidine) emerged from the column. Their elution times corresponded to the first three new peaks found in the hydrolysates of acrolein treated alpha 1-PI.

The inducibility of metallothionein by cis- and trans-diamminedichloroplatinum(II) and their hydrolyzed products was studied in the JAr (a human choriocarcinoma) cell line. The metallothionein present in the cytosolic fraction of these cells was measured after a 12- or 24-h exposure to a fresh metal solution or a solution that had been allowed to hydrolyze. These results demonstrate that hydrolysis of the cis isomer produces a species that is able to give a 374% increase in the cytosolic level of metallothionein after a 24-h exposure to 40 microM of the metal. Fresh cisplatin, on the other hand, gives a maximal increase of 118% under the same conditions. Transplatin and its hydrolyzed species did not induce metallothionein. These results may indicate that hydrolysis of cisplatin performs an important role in the metabolism and possibly the toxicity of this widely used agent for cancer chemotherapy.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, and both males and females of this strain are sensitive to chemical induction of liver tumors. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors, and such activated oncogenes frequently contain a particular point mutation. In light of indications that the transforming capacity of some oncogenes is directly related to the level of the gene product, we hypothesized that transcriptional control of Ha-ras, Ki-ras, and myc is compromised in B6C3F1 mouse liver tumors. A positive correlation has been established between gene expression and hypomethylation. Therefore, the methylation states of these genes were examined in spontaneous liver tumors and in tumors induced by two diverse hepatocarcinogens: phenobarbital and chloroform. Ha-ras was found to be hypomethylated in all tumors examined, whereas Ki-ras was sometimes hypomethylated; such hypomethylation might play a role in the promotion stage of carcinogenesis. The methylation state of myc was unaltered, although this gene appeared to be amplified in tumors. These results suggest that a component of the mechanism by which these oncogenes are activated in B6C3F1 mouse liver tumors involves loss of stringent control of expression, via hypomethylation of the ras oncogenes and, possibly, amplification of myc. These results support the assertion that tumors induced by different classes of carcinogens or arising spontaneously share common biochemical pathways of oncogene activation during tumorigenesis.

We have previously demonstrated that a number of metal salts have the capacity to inhibit the DNA repair process in human cells. In order to determine a role for non-protein thiols (TNPT) in this inhibition, we investigated repair of X-ray damage in metal-treated HeLa cells under normal conditions and conditions in which cellular thiols had been depleted by treatment with buthionine sulfoximine (BSO) and diethyl maleate (DEM). The combination reduced cellular TNPT by 92%, and cells so depleted became sensitized to X-ray-induced killing and exhibited retarded sealing of X-ray-induced DNA single-strand breaks. Thiol depletion also sensitized cells to the cytotoxicity of certain but not all metals tested. The sensitivity to copper was increased over 6000-fold, and significant enhancement of killing was also seen in cells treated with arsenic, lead, and mercury. Smaller effects were observed with cadmium and nickel, and sensitivity to manganese, magnesium, cobalt or zinc was not substantially altered. Enhanced sensitivity to X-ray killing was found in cells treated with nickel, cadmium, zinc, arsenic, and copper under conditions in which thiols were not limiting. In thiol-depleted cells, sensitivity was not further increased in the case of nickel and arsenic but at least additively affected for copper, mercury and zinc. X-Ray-induced single-strand break repair was retarded by treatment of cells with mercury, nickel, zinc, arsenic, and copper in thiol-normal cells. In thiol-depleted cells, repair inhibition by zinc, arsenic, and copper was nearly complete, while little additional effect on repair was seen following mercury and nickel treatment. An examination of the effects of brief metal treatment on cellular TNPT revealed that copper strongly decreased thiol levels whereas the other metals tested either had no effect on TNPT or reduced TNPT levels to no less than 48% under the conditions employed. No simple relationship appears to exist relating loss of cellular thiols and sensitivity of repair in the series of metals tested. Clear, although indirect, evidence exists, however, that sensitivity to X-rays is mediated through thiols and that the interaction of metals and thiols in the cell may be an important factor in modulating the response to irradiation.

To further understand the molecular nature of changes leading to chemically induced ouabain resistance in C3H/10T1/2 Cl 8 (10T1/2) cells, we isolated plasma membranes from wild-type and ouabain-resistant (Ouar) 10T1/2 cells and characterized (Na,K)-ATPase activity in the plasma membrane fraction. (Na+,K+)-ATPase enzyme activity in membrane fractions extracted from wild-type 10T1/2 cells was inhibited in a concentration-dependent manner by ouabain and was completely inhibited by 2.4 mM ouabain. Lineweaver-Burke and Eisenthal/Cornish-Bowden analysis indicated that the inhibition was uncompetitive. Ten to 45% of (Na+,K+)-ATPase enzyme activity extracted from three Ouar 10T1/2 cell lines cultured in 1 mM ouabain was resistant to 2.4 mM ouabain, depending on the cell line. Resistance of (Na+,K+)-ATPase activity in the plasma membrane fraction of Ouar cells to inhibition by ouabain and resistance of cultured Ouar cells to the cytotoxicity of ouabain occurred over similar concentrations of ouabain (0.1-3mM). Two ouabain-resistant cell lines, Ouar MNNG Cl 2 and Ouar MCA Cl 16-7, demonstrated the same total (Na+,K+)-ATPase specific activity as 10T1/2 cells, but the fraction of Ouar enzyme activity increased (from 18 to 40% in MNNG Cl 2 cells and from 10 to 25% in Sp Ouar Cl 16 cells) when the cells were cultured in ouabain. Thermal denaturation profiles and pH dependence profiles of (Na+,K+)-ATPase activity in plasma membranes from wild-type and Ouar 10T1/2 cells were identical. A 3.9-kb (Na,K)-ATPase alpha subunit mRNA transcript was found in 10T1/2 cells, and in the Ouar MNNG Cl 2 cell line cultured in the presence or absence of ouabain. There was no amplification of the gene coding for the alpha subunit of (Na+,K+)-ATPase in the chemically induced Ouar MNNG Cl 2 cell line, whether this cell line was cultured in the presence or absence of ouabain. These studies provide further evidence that the Ouar phenotype of chemically induced and spontaneous Ouar 10T1/2 cell lines derives from Ouar (Na+,K+)-ATPase activity, and that this Ouar (Na+,K+)-ATPase activity increases further in some cell lines cultured in the presence of ouabain.

Sister chromatid exchanges (SCE) have been examined in human lymphocytes following in vitro treatments with metal salts, nickel sulfate, lead sulfate and sodium arsenite. All of the metal salts produced significant increases in the SCE frequencies over the levels for untreated lymphocytes. The SCE frequencies were also examined for metal treatments combined with ultraviolet light (200 ergs/mm2). For the lead treatments combined with the UV dose selected, an additive SCE response was observed compared to the SCE responses for UV or metal alone. The nickel and arsenite treatments combined with UV produced a less than additive SCE response for most concentrations tested. These results suggest that nickel or arsenite present in complex mixtures may reduce the SCE response to other compounds in the mixture normally capable of producing a much stronger SCE response and therefore lead to an underestimate of the effects of chemical exposure.

We have previously shown that the inhibition of MNU-induced DNA repair by arsenite occurs after the incision step in Chinese hamster V79 cells. We now report that nuclear DNA ligase activity is inhibited after arsenite treatment and that the inhibited activity is mostly DNA ligase II. Both constitutive and MNU-inducible levels of DNA ligase II are inhibited. The addition of arsenite in vitro also indicates that DNA ligase II is more sensitive to arsenite inhibition than DNA ligase I. Since DNA ligase II is reported to be involved in the ligation step of excision repair, its inhibition by arsenite is a likely mechanism for the inhibition of DNA repair by arsenite and may account for the fact that arsenite acts as a comutagen with a number of different types of mutagens. The carcinogenicity of arsenite may also be a result of ligase inhibition.

The mutagenicity of a series of 19 aromatic amines had been previously measured in Salmonella typhimurium strains TA98 (frame-shift) and TA100 (base-pair) with the addition of S9 from Aroclor 1254-induced rat liver. A quantitative structure-activity relation (QSAR) study using multiple regression analysis points out the influence of three factors on mutagenicity: lipophilic character, position of the amine group, and whether it is free or acetylated, as expressed by log P and two indicator variables I1 and I2, respectively. The multiple regression equations explain 78 and 88% of the variance in log mutagenicity in TA98 and TA100, respectively. First of all, mutagenicity was shown to increase with lipophilicity. On the other hand, mutagenicity is reduced when the amine or acetamido position is ortho to the juncture because of steric hindrance in its biotransformation compared with a non-ortho isomer. It is decreased also by the acetylation of the amine group, probably because the acetyl group needs to be first split off prior to oxidation of the amine group to -NHOH.