Arsenic is widely distributed in the environment by natural and human means. The potential for adverse health effects from inorganic arsenic depends on the level and route of exposure. To estimate potential health risks of inorganic arsenic, the apportionment of exposure among sources of inorganic arsenic is critical. In this study, daily inorganic arsenic intake of U.S. adults from food, water, and soil ingestion and from airborne particle inhalation was estimated. To account for variations in exposure across the U.S., a Monte Carlo approach was taken using simulations for 100,000 individuals representing the age, gender, and county of residence of the U.S. population based on census data. Our analysis found that food is the greatest source of inorganic arsenic intake and that drinking water is the next highest contributor. Inhalation of airborne arsenic-containing particles and ingestion of arsenic-containing soils were negligible contributors. The exposure is best represented by the ranges of inorganic arsenic intake (at the 10th and 90th percentiles), which were 1.8 to 11.4 µg/day for males and 1.3 to 9.4 µg/day for females. Regional differences in inorganic arsenic exposure were due mostly to consumption of drinking water containing differing inorganic arsenic content rather than to food preferences.

The antitumor protein p53 plays a critical role in DNA repair. Inorganic arsenic exposure is associated with a wide variety of human tumors, particularly of the skin. To investigate how inorganic arsenic might interfere with DNA repair and lead to greater incidence of hyperkeratosis and skin tumors, we exposed human keratinocytes (HaCaT) to environmentally relevant concentrations of arsenite for 14 days. Arsenite reduced p53 levels while concomitantly increasing the p53 regulatory protein mdm2 levels in a dose- and time-dependent manner. We propose the disruption of the p53-mdm2 loop regulating cell cycle arrest as a model for arsenic-related skin carcinogenesis and it may be important in tumors with elevated mdm2 levels.

Arsenic is a ubiquitous contaminant of drinking water and food. The mechanisms of the toxic action of inorganic arsenic are unknown. We report the isolation of proteins having a high affinity for arsenic in the +3 oxidation state that are induced by arsenite (AsIII) in human lymphoblastoid cells. The arsenic-binding proteins were isolated using a p-aminophenylarsine oxide affinity column. At least four proteins of 50, 42, 38.5 and 19.5 kDa were isolated by elution with 10 or 100 mM 2-mercaptoethanol. Two proteins were tentatively identified as tubulin and actin on the basis of their molecular weights and previously reported affinity for the arsenic column. The identities of the remaining proteins are unknown. Heme oxygenase 1 was induced by AsIII but did not bind to the arsenic affinity column. We conclude that AsIII induces multiple proteins that have variable affinities for arsenic in the +3 state as judged by the concentration of 2-mercaptoethanol required for their elution. The arsenic binding motif of these proteins may involve three thiol groups arranged 3-6 A apart by the tertiary structure of the protein as suggested by others. These proteins may serve as high affinity binding sites for AsIII and may be involved in the biological action of AsIII.

Use of oxygenates in gasoline in the United States may increase atmospheric levels of aldehydes. To assist in health assessments of inhalation exposure to aldehydes, we studied glutathione (GSH) depletion by low-molecular-weight n-alkanals and 2-alkenals, ubiquitous air pollutants, in adult rat lung (ARL) cells by laser cytometry. For each homologous series, the effective aldehyde concentration that depleted GSH by 50% (EC50) in ARL cells correlates with published values for the median lethal dose of the chemicals and with Hammett/Taft electronic parameters, σ* for n-alkanals and σp* for 2-alkenals. n-Alkanals (EC50, 110–400 mmol/L) were 1000 times less effective in depleting GSH than were 2-alkenals (EC50, 2–180 μmol/L), of which acrolein was the most potent. Ability of the 2-alkenals to deplete GSH follows the second-order rate constant for adduct formation. Ability of n-alkanals to deplete GSH follows chain length. Within a homologous series of low-molecular-weight aldehydes, structure–activity relationships are useful for predicting the toxicity of the aldehydes in vitro and in vivo.

Dietary arsenic intake estimates based on surveys of total arsenic concentrations appear to be dominated by intake of the relatively non-toxic, organic arsenic forms found in seafood. Concentrations of inorganic arsenic in food have not been not well characterized. Accurate dietary intake estimates for inorganic arsenic are needed to support studies of arsenic's status as an essential nutrient, and to establish background levels of exposure to inorganic arsenic. In the market basket survey reported here, 40 commodities anticipated to provide at least 90% of dietary inorganic arsenic intake were identified. Four samples of each commodity were collected. Total arsenic was analysed using an NaOH digestion and inductively coupled plasma-mass spectrometry. Separate aliquots were analysed for arsenic species using an HCl digestion and hydride atomic absorption spectroscopy. Consistent with earlier studies, total arsenic concentrations (all concentrations reported as elemental arsenic per tissue wet weight) were highest in the seafoods sampled (ranging from 160 ng/g in freshwater fish to 2360 ng/g in saltwater fish). In contrast, average inorganic arsenic in seafood ranged from less than 1 ng/g to 2 ng/g. The highest inorganic arsenic values were found in raw rice (74 ng/g), followed by flour (11 ng/g), grape juice (9 ng/g) and cooked spinach (6 ng/g). Thus, grains and produce are expected to be significant contributors to dietary inorganic arsenic intake.

Background exposures to inorganic arsenic have been estimated to be in the range of 16-19 mu g/day with diet being the primary source. However, data on concentrations of inorganic arsenic in food are scanty. Existing literature includes data for a limited number of foods, and suggests that daily dietary inorganic arsenic intake for various age groups ranges from 8 to 14 mu g/day in the United States and from 5 to 13 mu g/day in Canada. The current study was conducted to obtain data for a larger number and variety of foods, and to more precisely estimate background dietary exposure ranges in the United States. This analysis presents results of a market basket survey of 40 commodities selected to represent samples of most major food types and to account for the majority of dietary exposure to inorganic arsenic. Samples were analyzed for total arsenic using NaOH digestion and inductively coupled plasma-mass spectrometry. Separate aliquots were analyzed for As3+, As5+, MMA, and DMA using an HCl digestion and hydride atomic absorption. The highest inorganic arsenic concentrations were found in grains and produce. Total arsenic concentrations were highest in seafood, and were consistent with results from previous studies; however, average inorganic arsenic concentrations in seafood were lower than previously reported. Data from the foods surveyed were coupled with a United States food consumption database to estimate the distribution of the dietary inorganic arsenic intake among adults. For most adults, dietary inorganic arsenic intake is predicted to fall within the range of 1-20 mu g/day.

Susceptibility to bladder or colorectal cancer in humans exposed to arylamines or heterocyclic amines may be influenced substantially by the activities of the polymorphic enzymes cytochrome P4501A2 (CYP1A2) and N-acetyltransferase (NAT). An association between colorectal cancer and CYP1A2 and NAT activities is controversial. CYP1A2 and NAT phenotypes were determined simultaneously using micellar electrokinetic chromatography of caffeine metabolites in urine extracts following a single oral dose of approximately 120 mg caffeine. The peak area ratio of 1,7-dimethylxanthine/ 1,3,7-trimethylxanthine (caffeine) was used for CYP1A2 phenotyping. The peak area ratio of 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine was used for NAT phenotyping. The NAT2* genotype was evaluated by restriction fragment length polymorphism analysis using capillary electrophoresis with a sieving buffer containing ethidium bromide. Capillary electrophoresis is a rapid and simple method that can be automated for CYP1A2 and NAT phenotyping by analysis of caffeine urinary metabolites as well as for NAT2* genotyping.

We propose the use of human lymphocyte heme oxygenase 1 (HO1) as a biomarker of response to environmental arsenic exposure. We report the induction of HO1 in human lymphoblastoid cells (LBs) by arsenite in a dose-related manner. HO1 was identified by SDS-PAGE from its molecular weight and from its detection by Western blotting with anti-HO1. HO1 levels in LBs treated with arsenite increased by de novo synthesis as demonstrated by incorporation of 35S-methionine and by inhibition of HO1 synthesis by actinomycin D. The amount of HO1 in LBs was estimated by quantifying Western blots. HO1 was also induced by 10 microM cadmium or mercuric chloride. We suggest that circulating lymphocyte HO1 levels may be useful in assessing the biological activity of arsenic exposure in vivo under properly controlled conditions of simultaneous urinalysis for arsenic, cadmium, and mercury.

This study was undertaken to compare the genotoxic effects of arsenite in cultured human lymphocytes and lymphoblastoid cell lines from a group of normal human volunteers. The goal was to determine whether, as found with other genotoxins, subgroups might exist which showed relative high or low sensitivity to induction of sister chromatid exchanges (SCEs) by this metal. Primary lymphoblast cultures were established by treatment with phytohemagglutinin (PHA-L). Lymphoblastoid cell lines were established by transformation with Epstein-Barr virus. Cultures were exposed for 40 h to sodium arsenite (AsIII) and SCEs assayed by 5-bromo-2′-deoxyuridine incorporation and staining by fluorescence plus Giemsa. SCEs were increased by arsenite in a dose-dependent manner over the concentration range of 10−7–10−5 M. SCEs could not be scored above 10−5 M because of cytotoxicity. Comparison of SCE frequency in primary lymphocyte cultures among individuals showed substantial variation in sensitivity to arsenite, with some showing no significant effect while others showed a 2–3-fold increase in SCE frequency. In one lymphoblastoid cell line especially sensitive to arsenite, arsenic acid (AsV) or dimethylarsinic acid (DMA) at concentrations up to 10−5 M did not increase the SCE frequency suggesting that AsIII is the active form of arsenic. When pooled data from the primary lymphocytes was compared to that obtained with the lymphoblastoid cells, the slopes of the dose-response curves for AsIII-induced SCEs were similar. The sensitivity of the majority of the individual primary lymphocyte cultures to SCE induction by arsenite was correlated with the sensitivity of the lymphoblastoid cultures established from the same individual. However, in three individuals no correlation was found. Individual lymphoblastoid cell lines retained their As sensitivity after cryopreservation and subsequent revival. Whether the genotoxic response to As is genetically controlled or the result of phenotypic selection is being explored in these stable lymphoblastoid cell lines.

The proceedings in this volume suggest several important future research needs. Research is needed to validate modeling in toxicology in order to support the use of the general approach of modeling, either for structure-activity relationships (SAR) or physiologically based pharmacokinetics (PBPK), by regulators. Research is needed to demonstrate how SAR predictions can be integrated with the PBPK models to reduce the chemical-specific, intensive data requirements of PBPK models. Methods are needed to harmonize modeling approaches and to educate potential users. New approaches to modeling are needed which go beyond present day assumptions of flow-limited models into dynamic models of human health effects. The academic community can further these objectives by including applied mathematics of toxicology, including both SAR and PBPK modeling, in toxicology curricula.

Nitrogen dioxide (NO2) and ozone (O3) occur throughout the world as the primary pollutants of urban air. NO2 and O3 oxidize cell membrane lipids and proteins. Inflammatory agents are elaborated from the lung either as a direct result of oxidation or as a consequence of leukocytes recruited into the lung by injury. My hypothesis is that NO2 and O3 initiate or exacerbate chronic lung disease through an inflammatory mechanism which can be reduced by supplementation with greater amounts than those required to alleviate vitamin deficiency symptoms of vitamins C (ascorbic acid) and E (α-tocopherol). Children, whose lungs are developing, are the most likely group to benefit from supplementation with vitamins C and E because the adverse effects of inflammation on the developing lung are likely to be greater and the time of exposure is longer than in adults. This hypothesis is in accord with current human and experimental animal data and the chemistry of O3 and NO2 toxicity, and is supported by recent ecological epidemiological studies of persons supplementing their intake of vitamins C and E.

Pulmonary macrophages (PM) play a key role in the immune defenses of the lung. When stimulated, PM express Fc receptors (FcR) that regulate the immune response. PM were assayed for FcR expression following subchronic inhalation exposure of adult Fischer 344 rats to either 90 μg/m3 nitrate (NH4NO3), 300 μg/m3 road dust, or clean air, for 4 h/day, 4 days/week, for 8 weeks. PM were lavaged from the lungs and attached to glass coverslips for 18 h. PM FcR were labelled with rat IgG conjugated with cyanine-3. For each exposure, FcR were determined with a Meridian ACAS 570 confocal cytometer by imaging the fluorescence of 50 cells. We found that the IgG binding to FcR (in arbitrary fluorescence units, FU, per cell) for PM from road dust exposed rats was less (835 ± 39.3 FU/cell) than that for PM from both ammonium nitrate or clean air-exposed rats (1115 ± 58.0 FU/cell and 1123 ± 46.6 FU/cell, respectively). While acid incubation conditions in vitro (pH 5.5 for 30 min to simulate the acid environment of ammonium nitrate inhalation) resulted in a 16% decrease in IgG binding (P < 0.05), IgG binding to PM from acid aerosol exposed rats was no different than the IgG bound to PM from clean air-exposed rats. PM exposed to road dust in vivo expressed 25% fewer FcR (P < 0.05). Three-dimensional images of PM failed to show any major alterations in FcR distribution. These preliminary results indicate cellular recognition of antibody-immune complexes may be impaired by subchronic exposure to road dust, which could decrease the immune response of road dust exposed animals.

Although the evidence for oxidative stress for air pollution in the human lung is fragmentary, the hypothesis that oxidative stress is an important, if not the sole, mechanism of toxicity of oxidizing air pollutants and tobacco smoke is compelling and growing. First, biochemical mechanisms have been worked out for oxidation of lung lipids by the gas phase of cigarette smoke, NO2 and O3. The oxidation of lung lipids can be prevented by both vitamins C and E. Vitamin C is more effective in preventing oxidation by NO2, and vitamin E is more effective against O3. Second, multiple species of experimental animals develop lung disease similar to human bronchitis and emphysema from exposure to NO2 and O3, respectively. The development of these diseases occurs over a near lifetime exposure when the levels of NO2 or O3 are at near ambient air pollution values. Third, isolated human cells are protected against oxidative damage from NO2 and O3 by both vitamins C and E. Fourth, the vitamin C level in the lung either declines on exposure to NO2 for short-term exposures or increases on chronic cigarette smoke exposure. The effects of cigarette smoking on serum vitamin C is apparently complex and may be related to the daily intake of vitamin C as well as smoking. Serum vitamin C levels may be poor indicators of lung demands when daily vitamin C intakes are above 100 mg/day. Fifth, vitamin C supplementation protects against the effects of ambient levels of air pollution in adults as measured by histamine challenge. An augmented response to histamine challenge may represent increased lung permeability brought about by air pollution. In experimental animal and human experiments, the amount of vitamin C or E that afforded protection was in excess of the current recommended dietary allowance. Although animal studies do not provide evidence for complete protection against NO2 or O3, they do illustrate that current recommended daily allowances are inadequate for maximum protection against air pollution levels to which over 100 million Americans are exposed. The problem of air pollution and its effects on humans is truly of global concern. Air pollution is not restricted to North America or Japan where it was first recognized, but is a major public health problem in Europe as well. When data are available, air pollution probably will be shown to be a major public health problem in all urban areas of the world.(ABSTRACT TRUNCATED AT 400 WORDS)

A mechanistic study was performed to elucidate the biochemical events connected with the cocarcinogenic effect of sulfur dioxide (SO2). Glutathione S-sulfonate (GSSO3H), a competitive inhibitor of the glutathione S-transferases, forms in lung cells exposed in culture to sulfite, the hydrated form of SO2. Changes in glutathione status (total GSH) were also observed during a 1-h exposure. Some cells were pretreated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione reductase. In human lung cells GSSO3H formed in a concentration-dependent manner, while glutathione (GSH) increased and glutathione disulfide (GSSG) decreased as the extracellular sulfite concentration was increased from 0 to 20 mM. The ratio of GSH/GSSG increased greater than 5-fold and the GSH/GSSO3H ratio decreased to 10 with increasing sulfite concentration. GSSO3H formed in rat lung cells exposed to sulfite, with no detectable effect on GSH and GSSG. GSSO3H also formed from cellular GSH mixed disulfides. GSSO3H formed rapidly, reaching its maximum value in 15 min. The viability of both cell types was unaffected except at 20 mM sulfite. GSSO3H incubated with human lung cells did not affect cellular viability. BCNU inhibited cellular GSSO3H reductase to the same extent as GSSG reductase. These results indicate that GSSO3H is formed in cells exposed to sulfite, and could be the active metabolite of sulfite responsible for the cocarcinogenic effect of SO2 by inhibiting conjugation of electrophiles by GSH.

This study seeks to characterize the uptake of hydroxyproline into the rat lung from the vascular system using the isolated ventilated perfused lung (IVPL) and further to determine whether this amine is trapped sufficiently to act as a reactant with NO2 in the production of N-nitrosohydroxyproline. Using blue dextran as an extracellular marker, hydroxyproline was found to be retained by the IVPL. The rate of hydroxyproline absorption by the lung from the perfusate did not increase linearly with hydroxyproline concentration but saturated at higher concentrations. This carrier-mediated hydroxyproline absorption had a Vmax of 968 nmol/min.g dry weight tissue and a Km of 217 μM. Similar to the 5-hydroxytryptamine uptake system, the uptake of hydroxyproline was inhibited in a sodium-free medium (66%). However, 5-hydroxytryptamine (or putrescine) did not inhibit uptake of hydroxyproline. Uptake of hydroxyproline was not sensitive to sodium cyanide or to carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. While this work shows that the lung preferentially sequesters hydroxyproline, published nitrosation rates of morpholine by NO2 suggest that only about 0.0001% hydroxyproline would be converted to nitrosohydroxyproline in the IVPL system. This uptake system represents a unique pulmonary carrier-mediated process in that it is unlike the 5-hydroxytryptamine or diamine pulmonary uptake systems previously described; it is a sodium-facilitated process, and the carrier-mediated uptake is not energy dependent. A rate of nitrosation sufficient to monitor by reaction of NO2 with radiolabeled hydroxyproline is unlikely to be obtained.

A general method is presented for the use of mathematical modeling in the design, execution, and interpretation of toxicology experiments. To illustrate the use of mathematical modeling toxicology, a case study is presented of how a dosimetry model for inhaled nickel was developed for use in cancer risk estimation. A physiologically based pharmacokinetic (PB-PK) dosimetry model is used to plan animal experiments and to extrapolate nickel kinetics from animals to humans. These data are then used to estimate human lung cancer risks from human exposure to nickel aerosols. To achieve this goal, a PB-PK dosimetry model for the lung was integrated with a PB-PK dosimetry model for the internal organs. Nickel removal from the lung was found to be saturable and to follow Michaelis-Menten kinetics. The PB-PK lung dosimetry model was used to design both short-term (single exposures) and long-term (multiple intermittent exposures) needed to validate the parameters (Km and Vmax) of the lung dosimetry model. A constant infusion experiment was planned using the PB-PK modeling approach to measure the distribution and elimination of intravenously administered nickel. The two PB-PK models were integrated to estimate the fate of nickel after inhalation and are being used to plan experiments for other routes of exposure such as ingestion of drinking water and dermal contact. The integrated model has been used to calculate a human cancer risk estimate in combination with short-term genotoxic experiments. Using PB-PK models in toxicology, as illustrated here, conserves experimental animals, aids in understanding new physiological phenomena (such as saturable clearance from the lung), incorporates in vitro tests with in vivo experiments, and provides a means of extrapolation to human health risks from multiple routes of exposure. Introducing the concepts of mathematical modeling into toxicity experiments at the beginning of the experiment improves the usefulness of the experiments in risk estimation. PB-PK models are suggested as a new basis for experimental design in toxicology.

In vivo O3 exposure followed by in vitro incubation was observed to cause inhibition of mouse RBC deformability. The requirement for in vitro incubation to allow expression of these effects and the potential role for oxidizable membrane components were investigated. Membrane sulfhydryls (SHs) and membrane ATPase are both susceptible to oxidation by O3 and are essential for maintaining RBC membrane deformability. RBC SH levels and ATPase activity were unchanged immediately after exposure of mice to filtered air (controls) or 1 ppm O3. After a subsequent 6-hr in vitro incubation, RBCs from control mice exhibited significant increases in membrane SH and ATPase activity, while SH levels and ATPase activity in RBCs from O3-exposed mice remained unchanged. Although the stimulus for increasing membrane SH and ATPase activity is unclear, these changes appear to be essential to maintaining RBC deformability in vitro and are inhibited by in vivo O3 exposure.