Raymond S. H. Yang

Persistent Tissue Kinetics and Redistribution of Nanoparticles, Quantum Dot 705, in Mice: ICP-MS Quantitative Assessment

Background Quantum dots (QDs) are autofluorescent semiconductor nanocrystals that can be used for in vivo biomedical imaging. However, we know little about their in vivo disposition and health consequences. Objectives We assessed the tissue disposition and pharmacokinetics of QD705 in mice. Methods We determined quantitatively the blood and tissue kinetics of QD705 in mice after single intravenous (iv) injection at the dose of 40 pmol for up to 28 days. Inductively coupled plasma–mass spectrometry (ICP-MS) measurement of cadmium was the primary method of quantification of QD705. Fluorescence light microscopy revealed the localization of QD705 in tissues. Results Plasma half-life of QD705 in mice was short (18.5 hr), but ICP-MS analyses revealed QD705 persisted and even continued to increase in the spleen, liver, and kidney 28 days after an iv dose. Considerable time-dependent redistribution from body mass to liver and kidney was apparent between 1 and 28 days postdosing. The recoveries at both time points were near 100%; all QD705s reside in the body. Neither fecal nor urinary excretion of QD705 was detected appreciably in 28 days postdosing. Fluorescence microscopy demonstrated deposition of QD705 in the liver, spleen, and kidneys. Conclusion Judging from the continued increase in the liver (29–42% of the administered dose), kidney (1.5–9.2%), and spleen (4.8–5.2%) between 1 and 28 days without any appreciable excretion, QD705 has a very long half-life, potentially weeks or even months, in the body and its health consequences deserve serious consideration.

Pharmacokinetics, metabolism, and carcinogenicity of arsenic.

The carcinogenicity of arsenic in humans has been unambiguously demonstrated in a variety of epidemiological studies encompassing geographically diverse study populations and multiple exposure scenarios. Despite the abundance of human data, our knowledge of the mechanism(s) responsible for the carcinogenic effects of arsenic remains incomplete. A deeper understanding of these mechanisms is highly dependent on the development of appropriate experimental models, both in vitro and in vivo, for future mechanistic investigations. Suitable in vitro models would facilitate further investigation of the critical chemical species (arsenate/arsenite/MMA/DMA) involved in the carcinogenic process, as well as the evaluation of the generation and role of ROS. Mechanisms underlying the clastogenic effects of arsenic, its role in modulating DNA methylation, and the phenomenon of inducible tolerance could all be more completely investigated using in vitro models. The mechanisms involved in arsenics inhibition of ubiquitin-mediated proteolysis demand further attention, particularly with respect to its effects on cell proliferation and DNA repair. Exploration of the mechanisms responsible for the protective or anticarcinogenic effects of arsenic could also enhance our understanding of the cellular and molecular interactions that influence its carcinogenicity. In addition, appropriate in vivo models must be developed that consider the action of arsenic as a promoter and/or progressor. In vivo models that allow further investigation of the comutagenic effects of arsenic are also especially necessary. Such models may employ initiation-promotion-progression bioassays or transgenic animals. Both in vitro and in vivo models have the potential to greatly enhance our current understanding of the cellular and molecular interactions of arsenic and its metabolites in target tissues. However, refinement of our knowledge of the mechanistic aspects of arsenic carcinogenicity is not alone sufficient; an understanding of the pharmacokinetics and target tissue doses of the critical chemical species is essential. Additionally, a more thorough characterization of species differences in the tissue kinetics of arsenic and its methylated metabolites would facilitate the development of more accurate and relevant PBPK models. Improved models could be used to further investigate the existence of a methylation threshold for arsenic and its relevance to arsenic carcinogenicity in humans. The significance of alterations in relative tissue concentrations of SAM and SAH deserves further attention, particularly with respect to their role in modulating methyltransferases involved in arsenic metabolism and DNA methylation. The importance of genetic polymorphisms and nutrition in influencing methyltransferase activities must not be overlooked. In vivo models are necessary to evaluate these factors; transgenic or knockout models would be particularly useful in the investigation of methylation polymorphisms. Further evaluation of methylation polymorphisms in human populations is also warranted. Other in vivo models incorporating dietary manipulation could provide valuable insight into the role of nutrition in the carcinogenicity of arsenic. With more complete knowledge of the pharmacokinetics of arsenic metabolism and the mechanisms associated with its carcinogenic effects, development of more reliable risk assessment strategies are possible. Integration of data, both pharmacokinetic and mechanistic in nature, will lead to more accurate descriptions of the interactions that occur between the active chemical species and cellular constituents which lead to the development of cancer. This knowledge, in turn, will facilitate the development of more accurate and reliable risk assessment strategies for arsenic.

Toxicological studies of chemical mixtures of environmental concern at the National Toxicology Program: Health effects of groundwater contaminants☆

In cooperation with the Agency for Toxic Substances and Disease Registry, the National Toxicology Program is participating in a Public Health Service activity related to the Comprehensive Environmental Response, Compensation and Liability Act (Superfund Act) by conducting toxicology studies on chemicals found in high-priority hazardous waste sites and for which adequate toxicological data are not available. As part of this effort, a project on the toxicology of chemical mixtures of groundwater contaminants was initiated. The first study, centered on the health effects of groundwater contaminants, is at the contractual stage. Nineteen organic and six inorganic chemicals, selected from more than 1000 known groundwater contaminants, will be given in drinking water to Fischer 344 rats and B6C3F1 mice for 3 or 6 months. Controls and five dose levels, based on average concentrations (i.e., baseline level) of individual component chemicals, or 0.1-, 10-, or 1000-fold of the baseline level, will be used. Toxicological end points include mortality, clinical signs, water and food consumption, body and organ weights, clinical pathology analytes (e.g., hematology, clinical chemistry, and urinalysis), gross and histopathology, neurobehavioral tests, sperm morphology and vaginal cytology evaluations (SMVCE), and cytogenetics. This paper summarizes the rationale behind our experimental design and the factors one must consider when designing studies of complex chemical mixtures.

Critical analysis of literature on low-dose synergy for use in screening chemical mixtures for risk assessment

There is increasing interest in the use of tiered approaches in risk assessment of mixtures or co-exposures to chemicals for prioritization. One possible screening-level risk assessment approach is the threshold of toxicological concern (TTC). To date, default assumptions of dose or response additivity have been used to characterize the toxicity of chemical mixtures. Before a screening-level approach could be used, it is essential to know whether synergistic interactions can occur at low, environmentally relevant exposure levels. Studies demonstrating synergism in mammalian test systems were identified from the literature, with emphasis on studies performed at doses close to the points of departure (PODs) for individual chemicals. This search identified 90 studies on mixtures. Few included quantitative estimates of low-dose synergy; calculations of the magnitude of interaction were included in only 11 papers. Quantitative methodology varied across studies in terms of the null hypothesis, response measured, POD used to test for synergy, and consideration of the slope of the dose-response curve. It was concluded that consistent approaches should be applied for quantification of synergy, including that synergy be defined in terms of departure from dose additivity; uniform procedures be developed for assessing synergy at low exposures; and the method for determining the POD for calculating synergy be standardized. After evaluation of the six studies that provided useful quantitative estimates of synergy, the magnitude of synergy at low doses did not exceed the levels predicted by additive models by more than a factor of 4.

Variability in Biological Exposure Indices Using Physiologically Based Pharmacokinetic Modeling and Monte Carlo Simulation

By using physiologically based pharmacokinetic (PBPK) modeling coupled with Monte Carlo simulation, the interindividual variability in the concentrations of chemicals in a workers exhaled breath and urine were estimated and compared with existing biological exposure indices (BEIs). The PBPK model simulated an exposure regimen similar to a typical workday, while exposure concentrations were set to equal the ambient threshold limit values (TLVs) of six industrial solvents (benzene, chloroform, carbon tetrachloride, methylene chloride, methyl chloroform, and trichloroethylene). Based on model predictions incorporating interindividual variability, the percentage of population protected was derived using TLVs as the basis for worker protection. Results showed that current BEIs may not protect the majority or all of the workers in an occupational setting. For instance, current end-expired air indices for benzene and methyl chloroform protect 95% and less than 10% of the worker population, respectively. Urinary metabolite concentrations for benzene, methyl chloroform, and trichloroethylene were also estimated. The current BEI recommendation for phenol metabolite concentration at the end-of-shift sampling interval was estimated to protect 68% of the worker population, while trichloroacetic acid (TCAA) and trichloroethanol (TCOH) concentrations for methyl chloroform exposure were estimated to protect 54% and 97%, respectively. The recommended concentration of TCAA in urine as a determinant of trichloroethylene exposure protects an estimated 84% of the workers. Although many of the existing BEIs considered appear to protect a majority of the worker population, an inconsistent proportion of the population is protected. The information presented in this study may provide a new approach for administrative decisions establishing BEIs and allow uniform application of biological monitoring among different chemicals.

Analyses of cytogenetic damage in rodents following exposure to simulated groundwater contaminated with pesticides and a fertilizer

Male Fischer 344 rats and female B6C3F1 mice were each exposed through their drinking water to a mixture of pesticides and ammonium nitrate that simulated contaminated groundwater in California (California Chemical Mixture [CCM]). Exposures were for 71 or 91 days, respectively. In addition, B6C3F1 female mice were exposed for 91 days to another pesticide and ammonium nitrate mixture (Iowa Chemical Mixture [ICM]) through their drinking water. The spleens were removed from the animals, and the splenocytes were cultured for analyses of sister-chromatid exchange (SCE), chromosome aberrations (CA), and micronuclei (MN) in cytochalasin B-induced binucleate cells. A concentration-related increase in SCEs was found in the splenocytes of the rat at the 1x, 10x and 100x levels of the CCM and at the 100x concentration of the CCM in the mouse. There were no other consistent cytogenetic effects observed with the CCM, and no statistically significant cytogenetic damage was observed in mice exposed to the ICM. Evidence from the literature is discussed in order to infer which chemical or chemicals in the CCM might be responsible for the observed SCE response.

Enzymatic Conjugation and Insecticide Metabolism

Present knowledge concerning biochemical conjugations stemmed from the isolation and identification of conjugates from normal and pathological animal urine during the last century. As early as 1842, the formation of hippuric acid was conclusively established by Keller following self-administration of benzoic acid. During the latter part of the last century, conjugations such as the synthesis of sulfates, glucuronides, and ornithine conjugates were discovered, and reactions involving mercapturic acid formation, methylation, acetylation, and cyanide detoxication were recognized. Glutamine conjugation was demonstrated in man in 1914 and glucoside conjugation in plants and insects in 1938 and 1953, respectively (Smith and Williams, 1970; Williams, 1959, 1967). During the past two decades, considerable attention has centred around the biochemical conjugations of certain naturally occurring body constituents such as sterols, bile acids, steroid hormones, glycoproteins, and mucopolysaccharides. Consequently, a number of new conjugations have been discovered (Layne, 1970).

Toxicology Studies of a Chemical Mixture of 25 Groundwater Contaminants II. Immunosuppression in B6C3F1 Mice

Concern over the potential adverse health effects of chemically contaminated groundwater has existed for many years. In general, these studies have focused on retrospective epidemiological studies for cancer risk. In the present studies, immune function was monitored in female B6C3F1 mice exposed to a chemical mixture in drinking water for either 14 or 90 days. The mixture consisted of 25 common groundwater contaminants frequently found near toxic waste dumps, as determined by EPA surveys. None of the animals developed overt signs of toxicity such as body or liver weight changes. Mice exposed to the highest dose of this mixture for 14 or 90 days showed immune function changes which could be related to rapidly proliferating cells, including suppression of hematopoietic stem cells and of antigen-induced antibody-forming cells. Some of these responses, e.g., granulocyte-macrophage colony formation, were also suppressed at lower concentrations of the chemical mixture. There were no effects on T cell function or T and B cell numbers in any of the treatment groups. Altered resistance to challenge with an infectious agent also occurred in mice given the highest concentration, which correlated with the immune function changes. Paired-water studies indicated that the immune effects were related to chemical exposure and not to decreased water intake. These results suggest that long-term exposure to contaminated groundwater may represent a risk to the immune system in humans.

Toxicology of chemical mixtures: experimental approaches, underlying concepts, and some results.

The toxicology of chemical mixtures will be the toxicology of the 1990s and beyond. While this branch of toxicology most closely reflects the actual human exposure situation, there is yet no standard protocol or consensus methodology for investigating the toxicology of mixtures. Thus, in this emerging science, experimentation is required just to develop a broadly applicable evaluation system. Several examples are discussed to illustrate the different experimental designs and the concepts behind each. These include the health effects studies of Love Canal soil samples, the Lake Ontario Coho salmon, the water samples repurified from secondary sewage in the city of Denver Potable Water Reuse Demonstration Plant, and the National Toxicology Program (NTP) effort on a mixture of 25 frequently detected groundwater contaminants derived from hazardous waste disposal sites. In the last instance, an extensive research program has been ongoing for the last 2 years at the NTP, encompassing general toxicology, immunotoxicology, developmental and reproductive toxicology, biochemical toxicology, myelotoxicology, genetic toxicology, neurobehavioral toxicology, and hepato- and renal toxicology.

Statistical analysis of interactive cytotoxicity in human epidermal keratinocytes following exposure to a mixture of four metals

Exposure to mixtures of chemicals is an important and relevant environmental issue. Of particular interest is the detection and characterization of departure of biological effects from additivity. Methodology based on the assumption of additivity is used in fitting single-chemicaldata. Interactionsare determined and characterized by making comparisons between the observed and predicted responses at mixtures along a fixed ratio ray of the component substances. Two simultaneous tests are developed for testing for any departure from additivity. Multiple comparisons procedures are used to compare observed responses to that predicted under additivity. A simultaneous confidence band on the predicted responses along the mixture ray is also developed. The methods are illustrated with cytotoxicity data that arise when human epidermal keratinocytes are exposed to a mixture of arsenic, chromium, cadmium, and lead. Synergistic, antagonistic, and additive cytotoxicities were observed at different dose levels of the four-metal mixture.