Chromate efflux by means of the ChrA chromate resistance protein from Pseudomonas aeruginosa.
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Everted membrane vesicles of Pseudomonas aeruginosa PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more (51)CrO(4)(2-) than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent K(m) of 0.12 mM chromate and a V(max) of 0. 5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential. (+info)
Mortality among aircraft manufacturing workers.
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OBJECTIVES: To evaluate the risk of cancer and other diseases among workers engaged in aircraft manufacturing and potentially exposed to compounds containing chromate, trichloroethylene (TCE), perchloroethylene (PCE), and mixed solvents. METHODS: A retrospective cohort mortality study was conducted of workers employed for at least 1 year at a large aircraft manufacturing facility in California on or after 1 January 1960. The mortality experience of these workers was determined by examination of national, state, and company records to the end of 1996. Standardised mortality ratios (SMRs) were evaluated comparing the observed numbers of deaths among workers with those expected in the general population adjusting for age, sex, race, and calendar year. The SMRs for 40 cause of death categories were computed for the total cohort and for subgroups defined by sex, race, position in the factory, work duration, year of first employment, latency, and broad occupational groups. Factory job titles were classified as to likely use of chemicals, and internal Poisson regression analyses were used to compute mortality risk ratios for categories of years of exposure to chromate, TCE, PCE, and mixed solvents, with unexposed factory workers serving as referents. RESULTS: The study cohort comprised 77,965 workers who accrued nearly 1.9 million person-years of follow up (mean 24.2 years). Mortality follow up, estimated as 99% complete, showed that 20,236 workers had died by 31 December 1996, with cause of death obtained for 98%. Workers experienced low overall mortality (all causes of death SMR 0.83) and low cancer mortality (SMR 0.90). No significant increases in risk were found for any of the 40 specific cause of death categories, whereas for several causes the numbers of deaths were significantly below expectation. Analyses by occupational group and specific job titles showed no remarkable mortality patterns. Factory workers estimated to have been routinely exposed to chromate were not at increased risk of total cancer (SMR 0.93) or of lung cancer (SMR 1.02). Workers routinely exposed to TCE, PCE, or a mixture of solvents also were not at increased risk of total cancer (SMRs 0.86, 1.07, and 0.89, respectively), and the numbers of deaths for specific cancer sites were close to expected values. Slight to moderately increased rates of non-Hodgkin's lymphoma were found among workers exposed to TCE or PCE, but none was significant. A significant increase in testicular cancer was found among those with exposure to mixed solvents, but the excess was based on only six deaths and could not be linked to any particular solvent or job activity. Internal cohort analyses showed no significant trends of increased risk for any cancer with increasing years of exposure to chromate or solvents. CONCLUSIONS: The results from this large scale cohort study of workers followed up for over 3 decades provide no clear evidence that occupational exposures at the aircraft manufacturing factory resulted in increases in the risk of death from cancer or other diseases. Our findings support previous studies of aircraft workers in which cancer risks were generally at or below expected levels. (+info)
Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase.
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Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction. (+info)
Apoptosis and P53 induction in human lung fibroblasts exposed to chromium (VI): effect of ascorbate and tocopherol.
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Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells. (+info)
Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis.
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The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO4(2-) inhibit the tubulin polymerization at an IC50 of 14-24 microM with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 microM. BIAcore measurements for tubulin-tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO4(2-). No inhibitory effect of Cu2+ or CrO4(2-) on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO4(2-) ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10-14 microM with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems. (+info)
Activation of JNK, p38 and ERK mitogen-activated protein kinases by chromium(VI) is mediated through oxidative stress but does not affect cytotoxicity.
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In this study we have explored the involvement of oxidative stress in Cr(VI)-induced JNK, p38 and ERK signaling pathways and their effects on Cr(VI) cytotoxicity in human non-small cell lung carcinoma CL3 cells. Exposure to K(2)Cr(2)O(7) markedly activated JNK and p38 and moderately activated ERK in a dose- (10-80 microM) and time-dependent (1-12 h) manner. The activated p38 decreased markedly and rapidly and the activated JNK decreased gradually when Cr(VI) was removed from the medium. Post-incubation of Cr(VI)-treated cells with H(2)O(2) increased the activities of JNK and p38, but not ERK. Co-administering Cr(VI) with 3-amino-1,2, 4-triazole (3AT), a catalase inhibitor, enhanced p38 activation, but did not influence JNK and ERK activation by Cr(VI). Conversely, co-administering Cr(VI) with mannitol, a hydroxyl radical scavenger and a Cr(V) chelator, reduced p38 activation and increased JNK and ERK activation by Cr(VI). These results indicate that p38 activation by Cr(VI) is positively correlated with oxidative stress, while JNK activity can be enhanced by either a quencher (mannitol) or activator (H(2)O(2)) of redox reactions in Cr(VI)-exposed CL3 cells. However, both 3AT and mannitol reduced the cytotoxicity of Cr(VI), but H(2)O(2) did not. The JNK activated by Cr(VI) was decreased (approximately 50%) by expression of a kinase-defective form of MKK7 (MKK7A) but not that of MKK4 (MKK4KR), suggesting that activation of JNK by Cr(VI) is mediated through MKK7. SB202190, a specific inhibitor of p38, markedly decreased JNK but did not change ERK activation by Cr(VI). PD98059, a specific inhibitor of ERK kinases MKK1/2, blocked ERK and p38 but did not alter JNK activation by Cr(VI). Neither the specific kinase inhibitors nor expression of MKK7A altered Cr(VI)-induced cytotoxicity. Together, these results suggest that activation of the JNK, p38 and ERK pathways by Cr(VI) is mediated through diverse redox mechanisms, yet their activation does not correlate with Cr(VI) cytotoxicity. (+info)
Cyclosporin A inhibits chromium(VI)-induced apoptosis and mitochondrial cytochrome c release and restores clonogenic survival in CHO cells.
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A variety of key events in the molecular apoptotic pathway involve the mitochondria. Cyclosporin A (csA) affects the mitochondria by inhibiting the mitochondrial permeability transition (MPT), thereby preventing disruption of the transmembrane potential. The role of the MPT in apoptosis is not fully understood, but inhibition of the MPT may prevent the release of mitochondrial caspase activators, such as cytochrome c (cyt c), into the cytosol. Certain hexavalent chromium [Cr(VI)] compounds are known occupational respiratory tract toxins and carcinogens. We have previously shown that these compounds induce apoptosis as a predominant mode of cell death and that this effect can be observed in cell culture using soluble Cr(VI). We show here that Cr(VI)-induced apoptosis in Chinese hamster ovary (CHO) cells involves disruption of mitochondrial stability. Using a cyt c-specific monoclonal antibody, we observed a dose-dependent release of mitochondrial cyt c in cytosolic extracts of CHO cells exposed to apoptogenic doses of sodium chromate. Co-treatment of these cells with csA inhibited the release of cyt c and abrogated Cr(VI)-induced apoptosis as determined by a reduction in internucleosomal DNA fragmentation. Co-treatment with csA also markedly increased clonogenic survival of Cr(VI)-treated CHO cells. In contrast, the general caspase inhibitor Z-VAD-FMK markedly inhibited most of the morphological and biochemical parameters of apoptosis but did not prevent cyt c release and did not increase clonogenic survival. These results suggest that the MPT plays an important role in the regulation of mitochondrial cyt c release and that this may be a critical point in the apoptotic pathway in which cells are irreversibly committed to death. (+info)
Chromate reduction by a pseudomonad isolated from a site contaminated with chromated copper arsenate.
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A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter(-1) and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K(m) of 23 mg liter(-1) (437 microM) and a V(max) of 0.98 mg of Cr h(-1) mg of protein(-1) (317 nmol min(-1) mg of protein(-1)). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments. (+info)