Regine Goth-Goldstein

Particle-induced artifacts in the MTT and LDH viability assays

In vitro testing is a common first step in assessing combustion-generated and engineered nanoparticle-related health hazards. Commercially available viability assays are frequently used to compare the toxicity of different particle types and to generate dose-response data. Nanoparticles, well-known for having large surface areas and chemically active surfaces, may interfere with viability assays, producing a false assessment of toxicity and making it difficult to compare toxicity data. The objective of this study is to measure the extent of particle interference in two common viability assays, the MTT reduction and the lactate dehydrogenase (LDH) release assays. Diesel particles, activated carbon, flame soot, oxidized flame soot, and titanium dioxide particles are assessed for interactions with the MTT and LDH assay under cell-free conditions. Diesel particles, at concentrations as low as 0.05 μg/mL, reduce MTT. Other particle types reduce MTT only at a concentration of 50 μg/mL and higher. The activated carbon, soot, and oxidized soot particles bind LDH to varying extents, reducing the concentration measured in the LDH assay. The interfering effects of the particles explain in part the different toxicities measured in human bronchial epithelial cells (16HBE14o). We conclude that valid particle toxicity assessments can only be assured after first performing controls to verify that the particles under investigation do not interfere with a specific assay at the expected concentrations.

Cellular response to diesel exhaust particles strongly depends on the exposure method

In vitro exposure to aerosols at the air-liquid interface (ALI) preserves the physical and chemical characteristics of aerosol particles. Although frequently described as being a more physiologic exposure method, ALI exposure has not been directly compared with conventional in vitro exposures where the particles are suspended in medium. We exposed immortalized human bronchial epithelial cells (16HBE14o) to aerosolized diesel exhaust particles at the ALI and to suspensions of collected particles. The response of the cells was determined from measurements of the cell viability and interleukin-8 (IL-8) secretion. The deposited size distribution at the cell surface was measured with transmission electron microscopy to obtain a dose for the ALI exposure. Although exposure by either method caused a slight decrease in cell viability and induced IL-8 secretion, the response to ALI exposure occurred at doses several orders of magnitude lower than exposure to particles in suspension. The most likely sources for the different dose responses are the artifacts introduced during the collection and resuspension of particles for conventional suspension exposures. The number concentration of particles deposited at the ALI is similar to the modeled deposition in the tracheal-bronchial region in a human lung, but the ALI size distribution is skewed toward particles larger than those deposited in the lung.

Ethylnitrosourea-induced mutagenesis in asynchronous and synchronous Chinese hamster ovary cells

The toxic and mutagenic activity of the alkylating carcinogen N-ethyl-N-nitrosourea (ENU) was studied in Chinese hamster ovary (CHO) cells. Cell killing and induction of 6-thioguanine-resistant (TGr) and ouabain-resistant (OUAr) mutants were determined as a function of ENU dose and treatment time in asynchronous cell populations. A dose-dependent induction of mutants was observed. The mutation frequency did not increase with longer than 30-min treatment times, implying that ENU breaks down rapidly in the cell. When synchronous populations of CHO cells obtained by mitotic detachment were treated with ENU at various times during the cell cycle, ENU-induced reproductive death was strongly dependent on the position in the cell cycle at the time of treatment, the time of highest sensitivity being the beginning of the S period. The pattern of mutation induction by ENU over the cell cycle was quite different from the pattern for cell killing. The induction of TGr mutants seemed to be independent of cell-cycle time. The induction of OUAr mutants was also independent of cell-cycle time after a low ENU dose; however, after a high ENU dose the frequency of OUAr mutants varied during the cell cycle, with a slight enhancement in B1 and a decrease in the early S period. There was no sign of enhanced mutation induction at the growing point for the two genetic markers tested.

Distribution and accumulation of a mixture of arsenic, cadmium, chromium, nickel, and vanadium in mouse small intestine, kidneys, pancreas, and femur following oral administration in water or feed.

Manufactured gas plant (MGP) sites are contaminated with coal tar and may contain metals such as arsenic (As), cadmium (Cd), chromium (Cr), nickel (Ni), and vanadium (V). These metals are known to cause cancer or other adverse health conditions in humans, and the extent and cost of remediating MGP sites may be influenced by the presence of these metals. Studies assessed the distribution of these metals in female B6C3F1 mice ingesting (1) a metal mixture in water or (2) an MGP mixture in NIH-31 feed. The highest metal levels were measured in the small intestine and kidneys of mice receiving the metal mixture in water. For mice receiving the metal mixture in water, levels of As, Cd, and Cr, in the small intestine, levels of As, Cd, Cr, and V in the kidneys, levels of As and Cd in the pancreas, and levels of Cr and V in the femur were significantly greater than controls at 4, 8, 12, 16, and 24 wk. Except for Ni levels in the small intestine and femur and Cr levels in the kidneys, levels of metals were much lower in mice administered the MGP mixture in feed. The highest concentrations of metals in mice ingesting the MGP mixture in feed were found in the small intestine and kidneys, but few were significantly greater than controls. Levels of As in the small intestine at 6 and 18 wk and levels of Cr in the kidneys at 12, 18, and 24 wk were significantly greater than in controls. The data suggest that tissue burdens in small intestine, kidneys, pancreas, and femur of arsenic, cadmium, chromium, and vanadium are less when metals are present as an MGP mixture in feed than as a mixture in water. The reduced distribution and accumulation of metals in the organs of mice ingesting the MGP mixture in feed compared to the levels in organs of mice ingesting the metal mixture in water suggests that metals may be less likely to accumulate in humans ingesting MGP mixtures, thereby presenting a lower overall human health risk. The data presented indicate that the matrix in which metals are present will affect the uptake of individual metals and the organ specificity.

Individual human cell responses to low doses of chemicals studied by synchrotron infrared spectromicroscopy

Vibrational spectroscopy, when combined with synchrotron radiation-based (SR) microscopy, is a powerful new analytical tool with high spatial resolution for detecting biochemical changes in the individual living cells. In contrast to other microscopy methods that require fixing, drying, staining or labeling, SR-FTIR microscopy probes intact living cells providing a composite view of all of the molecular response and the ability to monitor the response over time in the same cell. Observed spectral changes include all types of lesions induced in that cell as well as cellular responses to external and internal stresses. These spectral changes combined with other analytical tools may provide a fundamental understanding of the key molecular mechanisms induced in response to stresses created by low- doses of chemicals. In this study we used the high spatial - resolution SR-FTIR vibrational spectromicroscopy as a sensitive analytical tool to detect chemical- and radiation- induced changes in individual human cells. Our preliminary spectral measurements indicate that this technique is sensitive enough to detect changes in nucleic acids and proteins of cells treated with environmentally relevant concentrations of dioxin. This technique has the potential to distinguish changes from exogenous or endogenous oxidative processes. Future development of this technique will allow rapid monitoring of cellular processes such as drug metabolism, early detection of disease, bio- compatibility of implant materials, cellular repair mechanisms, self assembly of cellular apparatus, cell differentiation and fetal development.

Cytochrome P4501B1 Expression in Normal Breast Tissue

Polycyclic aromatic hydrocarbons (PAHs) are metabolically activated to ultimate carcinogens by the cytochrome P-450 isozymes CYP1A1 and CYP1B1. High levels of these enzymes may result in increased DNA adduct formation and cancer initiation. We investigated whether expression of CYP1B1 in breast tissue varies to a similar extent as has been shown for CYP1A1 and whether increased CYP1B1 expression could constitute a risk factor for breast cancer. Expression of CYP1B1 and CYP1A1 was measured in a collection of 75 nontumor epithelial breast tissue specimens from breast cancer patients ( n = 36) and from cancer-free individuals ( n = 39). Using a semiquantitative reverse transcription (RT)-polymerase chain reaction (PCR) assay, CYP1B1 and CYP1A1 expression levels relative to the constantly expressed g - actin gene were determined. In this study, we found 300-fold and 1,000-fold interindividual variation in expression for CYP1B1 and CYP1A1 , respectively. The mean CYP1B1 transcript level in normal breast tissue was 70% higher in mastectomy patients compared with cancer-free individuals ( p = .0473). These data suggest that CYP1B1 may play a role in breast cancer etiology, particularly in women exposed to high levels of CYP1B1 substrates such as PAHs.

Detecting exposure to environmental organic toxins in individual cells: towards development of a micro-fabricated device

A new method is being developed for quickly screen for the human exposure potential to polycyclic aromatic hydrocarbons (PAHs) and organochlorines (OCs). The development involves two key elements: identifying suitable signals that represent intracellular changes that are specific to PAH and OC exposure, and constructing a device to guide the biological cell growth so that signals from individual cells are consistent and reproducible. We are completing the identification of suitable signals by using synchrotron radiation-based (SR) Fourier-transform infrared (FTIR) spectromicroscopy in the mid-infrared region (4000 - 400 cm-1). Distinct changes have been observed in the IR spectra after treatment of human cells in culture medium with PAHs and OCs. The potential use of this method for detecting exposure to PAHs and OCs has been tested and compared to a reverse transcription polymerase chain reaction (RT-PCR) assay that quantifies increased expression of the CYP1A1 gene in response to exposure to PAHs or OCs.

Role of CYP1B1 in PAH-DNA adduct formation and breast cancer risk

This study investigated the hypothesis that increased exposure to polycyclic aromatic hydrocarbons (PAHs) increases breast cancer risk. PAHs are products of incomplete burning of organic matter and are present in cigarette smoke, ambient air, drinking water, and diet. PAHs require metabolic transformation to bind to DNA, causing DNA adducts, which can lead to mutations and are thought to be an important pre-cancer marker. In breast tissue, PAHs appear to be metabolized to their cancer-causing form primarily by the cytochrome P450 enzyme CYP1B1. Because the genotoxic impact of PAH depends on their metabolism, we hypothesized that high CYP1B1 enzyme levels result in increased formation of PAH-DNA adducts in breast tissue, leading to increased development of breast cancer. We have investigated molecular mechanisms of the relationship between PAH exposure, CYP1B1 expression and breast cancer risk in a clinic-based case-control study. We collected histologically normal breast tissue from 56 women (43 cases and 13 controls) undergoing breast surgery and analyzed these specimens for CYP1B1 genotype, PAH-DNA adducts and CYP1B1 gene expression. We did not detect any difference in aromatic DNA adduct levels of cases and controls, only between smokers and non-smokers. CYP1B1 transcript levels were slightly lower in controls than cases, but the difference was not statistically significant. We found no correlation between the levels of CYP1B1 expression and DNA adducts. If CYP1B1 has any role in breast cancer etiology it might be through its metabolism of estrogen rather than its metabolism of PAHs. However, due to the lack of statistical power these results should be interpreted with caution.

Repair of alkylation damage in Saccharomyces cerevisiae

SummaryRepair of methylated bases in Saccharomyces cerevisiae was measured by two methods: in vitro in cell extracts, and in vivo, by determining the loss of methylated bases from yeast DNA after treatment of stationary cultures with [3H]-N-methyl-N′-nitro-N-nitrosoguanidine. Whereas no repair activity could be detected by the in vitro method, the methylated bases were removed in vivo very efficiently. These contradictory results of in vitro and in vivo repair measurements suggest that either the repair enzymes of yeast are sufficiently different from those of bacteria and mammalian cells that they are not active in the in vitro assay, or that methylated bases are repaired in yeast by a different pathway.