Sensitive quantitation of chromium-DNA adducts by inductively coupled plasma mass spectrometry with a direct injection high-efficiency nebulizer.

A novel method is described for the sensitive detection of chromium-DNA adducts. Chromium-DNA adducts were determined in 1 microgram of DNA from normal human lung fibroblasts exposed to sodium chromate using microscale flow injection analysis with a direct injection high-efficiency nebulizer and inductively coupled plasma mass spectrometry detection. The frequency of Cr-DNA adducts increased in a dose-dependent sigmoidal manner, indicating saturation and toxicity. The low detection limits (on the order of parts per trillion) allows the detection of as few as two Cr adducts per 10,000 bases, which, coupled with the small DNA sample requirement, makes this technique suitable for measuring metal-DNA adducts as biomarkers of exposure to toxic and carcinogenic metals such as Cr, in cultured cells, animals, and humans.

Apoptosis and P53 induction in human lung fibroblasts exposed to chromium (VI): effect of ascorbate and tocopherol.

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.

Chromium-induced genotoxicity and apoptosis: relationship to chromium carcinogenesis (review).

The adverse health effects linked with chromium (Cr) exposure, the role of solubility and chemical speciation of Cr compounds, and the diverse cellular and molecular effects of Cr make the study of Cr carcinogenesis and toxicology very interesting and complex. Certain Cr compounds are prominent metal carcinogens in both occupational and environmental settings. Inhaled particulate forms of hexavalent Cr [Cr(VI)] cause lung cancer as well as lung toxicity. Some of the important factors in determining the biological outcome of Cr exposure include the bioavailability, chemical speciation and solubility of Cr compounds, intracellular reduction, and interaction of Cr with DNA. The stable oxidation states of Cr found in nature are Cr(III) and Cr(VI). Cr(III) is unable to enter cells but Cr(VI) enters into cells through membrane anionic transporters. Intracellular Cr(VI) is metabolically reduced to the ultimate Cr(III). Cr(VI) does not react with macromolecules such as DNA, RNA, proteins and lipids. However, both Cr(III) and the reductional intermediate Cr(V) are capable of co-ordinate covalent interactions with macromolecules. At the genomic level, Cr genotoxicity manifests as gene mutations, several types of DNA lesions and inhibition of macromolecular synthesis. At the cellular level, Cr exposure may lead to cell cycle arrest, apoptosis, premature terminal growth arrest, or neoplastic transformation. Cr-induced DNA-DNA interstrand crosslinks (DDC), the tumor suppressor gene p53 and oxidative processes are some of the major factors that may play a significant role in determining the cellular outcome in response to Cr exposure. We have utilized cellular, molecular, pharmacological, and genetic approaches to understand the interrelationship between Cr-induced genotoxicity, apoptosis and carcinogenesis. This review is based on the results and inferences of this research. We hope this review will clarify existing concepts and also introduce novel perspectives in chromium carcinogenesis research.

Recirculating atomic beam oven.

We describe an atomic beam oven that reuses alkali metal in the unused portion of the beam. This is accomplished by condensing the desired vapor on a cooled jacket from which it may be drained back into the oven.

Coagulase-positive staphylococcal mastitis in a herd with low somatic cell counts.

An increase in clinical mastitis infections was observed in a high-producing 77-cow Holstein herd. Low bulk tank somatic cell counts and individual cow Dairy Herd Improvement Association somatic cell counts observed before, during, and after the epizootic were suggestive of herd environmental mastitis. However, bacteriologic culture survey of the total herd indicated that, in addition to infections possibly attributable to environmental pathogens, 22% (17/77) of the cows were infected with coagulase-positive Staphylococcus spp. Conceivably, investigative efforts and management changes, without bacteriologic culturing, might have resulted in reduction of the clinical infection rate in this herd. However, the continued high prevalence of a contagious pathogen with potential future implications would have gone unnoticed. Somatic cell count in milk from individual cows generally is a useful tool for monitoring the probability of intramammary infection, but must be complemented with bacteriologic culture of milk to determine whether contagious or environmental pathogens are responsible.

Costs associated with selected preventive practices and with episodes of clinical mastitis in nine herds with low somatic cell counts.

Nine dairy herds (mean size, 149 cows) with bulk-tank milk somatic cell counts of less than 300,000 cells/ml and greater than 80% of cows with Dairy Herd Improvement Association linear somatic cell counts less than or equal to 4 were selected for study. Each herd was monitored for 12 consecutive months. Duplicate quarter-milk specimens were collected from each cow for bacteriologic culturing at beginning of lactation, cessation of lactation, and at the time of each clinical episode of mastitis. Streptococcus agalactiae was never isolated and Staphylococcus aureus was isolated from less than 1% of all quarters. There were 554 episodes of clinical mastitis. During the year of study, the incidence rate of clinical mastitis varied from 15.6 to 63.7% of cows among the 9 herds. Mean costs per cow per year in herd for mastitis prevention were: $10 for paper towels, $3 for nonlactating cow treatment, and $10 for teat disinfectants. Mean cost associated with clinical mastitis was $107/episode. Approximately 84% ($90) of the costs attributed to a clinical episode were associated with decreased milk production and nonsalable milk. Costs of medication and professional veterinary fees per clinical episode varied significantly among the 9 herds. Three of the herds did not have a veterinarian treat a clinical episode of mastitis during the year of study even though 2 of these herds had the first and third highest incidence rates of clinical mastitis. When calculated on a per cow in herd basis, mean costs of $40/cow/year were attributed to clinical mastitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Matter-wave interferometry with phase fluctuating Bose-Einstein condensates.

Elongated Bose-Einstein condensates (BECs) exhibit strong spatial phase fluctuations even well below the BEC transition temperature. We demonstrate that atom interferometers using such condensates are robust against phase fluctuations; i.e., the relative phase of the split condensate is reproducible despite axial phase fluctuations. However, larger phase fluctuations limit the coherence time, especially in the presence of some asymmetries in the two wells of the interferometer.

Long phase coherence time and number squeezing of two Bose-Einstein condensates on an atom chip.

We measure the relative phase of two Bose-Einstein condensates confined in a radio frequency induced double-well potential on an atom chip. We observe phase coherence between the separated condensates for times up to approximately 200 ms after splitting, a factor of 10 longer than the phase diffusion time expected for a coherent state for our experimental conditions. The enhanced coherence time is attributed to number squeezing of the initial state by a factor of 10. In addition, we demonstrate a rotationally sensitive (Sagnac) geometry for a guided atom interferometer by propagating the split condensates.

Phase-sensitive recombination of two bose-einstein condensates on an atom chip.

The recombination of two split Bose-Einstein condensates on an atom chip is shown to result in heating which depends on the relative phase of the two condensates. This heating reduces the number of condensate atoms between 10% and 40% and provides a robust way to read out the phase of an atom interferometer without the need for ballistic expansion. The heating may be caused by the dissipation of dark solitons created during the merging of the condensates.

Low velocity quantum reflection of Bose-Einstein condensates.

We study how interactions affect the quantum reflection of Bose-Einstein condensates. A patterned silicon surface with a square array of pillars resulted in high reflection probabilities. For incident velocities greater than 2.5 mm/s, our observations agreed with single-particle theory. At velocities below 2.5 mm/s, the measured reflection probability saturated near 60% rather than increasing towards unity as predicted by the accepted theoretical model. We extend the theory of quantum reflection to account for the mean-field interactions of a condensate which suppresses quantum reflection at low velocity. The reflected condensates show collective excitations as recently predicted.

Light scattering to determine the relative phase of two Bose-Einstein condensates.

We demonstrated an experimental technique based on stimulated light scattering to continuously sample the relative phase of two spatially separated Bose-Einstein condensates of atoms. The phase measurement process created a relative phase between two condensates with no initial phase relation, read out the phase, and monitored the phase evolution. This technique was used to realize interferometry between two trapped Bose-Einstein condensates without need for splitting or recombining the atom cloud.

Optical weak link between two spatially separated Bose-Einstein condensates.

Two spatially separate Bose-Einstein condensates were prepared in an optical double-well potential. A bidirectional coupling between the two condensates was established by two pairs of Bragg beams which continuously outcoupled atoms in opposite directions. The atomic currents induced by the optical coupling depend on the relative phase of the two condensates and on an additional controllable coupling phase. This was observed through symmetric and antisymmetric correlations between the two outcoupled atom fluxes. A Josephson optical coupling of two condensates in a ring geometry is proposed. The continuous outcoupling method was used to monitor slow relative motions of two elongated condensates and characterize the trapping potential.

Contrast interferometry using Bose-Einstein condensates to measure h/m and alpha.

The kinetic energy of an atom recoiling due to absorption of a photon was measured as a frequency, using an interferometric technique called "contrast interferometry." Optical standing wave pulses were used to create a symmetric three-path interferometer with a Bose-Einstein condensate. Its recoil phase, measurable with a single shot, varies quadratically with additional recoils and is insensitive to errors from vibrations and ac Stark shifts. We have measured the photon recoil frequency of sodium to 7 ppm precision, using a simple realization of this scheme. Plausible extensions should yield sufficient precision to attain a ppb-level determination of h/m and the fine structure constant alpha.

Coreless vortex formation in a spinor Bose-Einstein condensate.

Coreless vortices were phase imprinted in a spinor Bose-Einstein condensate. The three-component order parameter of F=1 sodium condensates held in a Ioffe-Pritchard magnetic trap was manipulated by adiabatically reducing the magnetic bias field along the trap axis to zero. This distributed the condensate population across its three spin states and created a spin texture. Each spin state acquired a different phase winding which caused the spin components to separate radially.

From single- to multiple-pPhoton decoherence in an atom interferometer.

We measure the decoherence of a spatially separated atomic superposition due to spontaneous photon scattering. We observe a qualitative change in decoherence versus separation as the number of scattered photons increases, and verify quantitatively the decoherence rate constant in the many-photon limit. Our results illustrate an evolution of decoherence consistent with general models developed for a broad class of decoherence phenomena.

Chromium(VI) induces p53-dependent apoptosis in diploid human lung and mouse dermal fibroblasts.

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory-tract tissue and DNA and are thought to be human lung carcinogens. In general, Cr(VI) is mutagenic and carcinogenic at doses that also evoke some cell death, and we previously showed that the predominant mode of death is apoptosis. Because p53 has been shown to initiate apoptosis after genotoxic insults, the objective of these experiments was to determine whether p53 is activated in and necessary for apoptosis of normal diploid human lung fibroblasts (HLF cells) after chromium exposure. By using annexin(V) staining and fluorescent microscopy, we found that Cr(VI) caused up to 14% of HLF cells to undergo apoptosis within 24 h after exposure. In addition, by using western blotting, we found that p53 protein levels increased fourfold to sixfold after exposure to sodium chromate. Because the major function of p53 is as a transcription factor, it must be translocated from the cytoplasm to the nucleus after chromate exposure to be active. Immunofluorescence studies using an antibody against p53 showed that, after chromate exposure, p53 was located in the nucleus of the treated HLF cells. The necessity of p53 for chromium-induced apoptosis was examined in two ways. One approach used dermal fibroblasts from p53 wild-type, heterozygous, and null mice, and the other approach used HLF cells that were transiently transfected with the human papilloma virus E6 gene, which targets p53 for degradation and creates a functional p53-null cell. These studies showed that chromium-induced apoptosis was p53 dependent. Mol. Carcinog. 28:111-118, 2000.

Cyclosporin A inhibits chromium(VI)-induced apoptosis and mitochondrial cytochrome c release and restores clonogenic survival in CHO cells.

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.

Transport of Bose-Einstein condensates with optical tweezers.

We have transported gaseous Bose-Einstein condensates over distances up to 44 cm. This was accomplished by trapping the condensate in the focus of an infrared laser and translating the location of the laser focus with controlled acceleration. Condensates of order 10(6) atoms were moved into an auxiliary chamber and loaded into a magnetic trap formed by a Z-shaped wire. This transport technique avoids the optical and mechanical access constraints of conventional condensate experiments and creates many new scientific opportunities.