Immunization of Mice with a Live Transconjugant Shigella Hybrid Strain Induced Th1 and Th17 Cell-Mediated Immune Responses and Confirmed Passive Protection Against Heterologous Shigellae.

An avirulent, live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed in our earlier study by introducing a plasmid vector, pPR1347, into a Shiga toxin gene deleted Shigella dysenteriae 1. Three successive oral administrations of LTSHΔstx to female adult mice produced comprehensive passive heterologous protection in their offspring against challenge with wild-type shigellae. Production of NO and different cytokines such asIL-12p70, IL-1ß and IL-23 in peritoneal mice macrophages indicated that LTSHΔstx induced innate and adaptive immunity in mice. Furthermore, production of IFN-γ, IL-10 and IL-17 in LTSH-primed splenic CD4+ T cell suggested that LTSHΔstx may induce Th1 and Th17 cell-mediated immune responses. Exponential increase of the serum IgG and IgA titre against whole shigellae was observed in immunized adult mice during and after the immunization with the highest peak on day 35. Antigen-specific sIgA was also determined from intestinal lavage of immunized mice. The stomach extracts of neonates from immunized mice, mainly containing mother's milk, contained significant levels of anti-LTSHΔstx immunoglobulin. These studies suggest that the LTSHΔstx could be a new live oral vaccine candidate against shigellosis in the near future.

Economic-based projections of future land use in the conterminous United States under alternative policy scenarios.

Land-use change significantly contributes to biodiversity loss, invasive species spread, changes in biogeochemical cycles, and the loss of ecosystem services. Planning for a sustainable future requires a thorough understanding of expected land use at the fine spatial scales relevant for modeling many ecological processes and at dimensions appropriate for regional or national-level policy making. Our goal was to construct and parameterize an econometric model of land-use change to project future land use to the year 2051 at a fine spatial scale across the conterminous United States under several alternative land-use policy scenarios. We parameterized the econometric model of land-use change with the National Resource Inventory (NRI) 1992 and 1997 land-use data for 844 000 sample points. Land-use transitions were estimated for five land-use classes (cropland, pasture, range, forest, and urban). We predicted land-use change under four scenarios: business-as-usual, afforestation, removal of agricultural subsidies, and increased urban rents. Our results for the business-as-usual scenario showed widespread changes in land use, affecting 36% of the land area of the conterminous United States, with large increases in urban land (79%) and forest (7%), and declines in cropland (-16%) and pasture (-13%). Areas with particularly high rates of land-use change included the larger Chicago area, parts of the Pacific Northwest, and the Central Valley of California. However, while land-use change was substantial, differences in results among the four scenarios were relatively minor. The only scenario that was markedly different was the afforestation scenario, which resulted in an increase of forest area that was twice as high as the business-as-usual scenario. Land-use policies can affect trends, but only so much. The basic economic and demographic factors shaping land-use changes in the United States are powerful, and even fairly dramatic policy changes, showed only moderate deviations from the business-as-usual scenario. Given the magnitude of predicted land-use change, any attempts to identify a sustainable future or to predict the effects of climate change will have to take likely land-use changes into account. Econometric models that can simulate land-use change for broad areas with fine resolution are necessary to predict trends in ecosystem service provision and biodiversity persistence.

Charged tmRNA but not tmRNA-mediated proteolysis is essential for Neisseria gonorrhoeae viability.

tmRNA, through its tRNA and mRNA properties, adds short peptide tags to abnormal proteins, targeting these proteins for proteolytic degradation. Although the conservation of tmRNA throughout the bacterial kingdom suggests that it must provide a strong selective advantage, it has not been shown to be essential for any bacterium. We report that tmRNA is essential in Neisseria gonorrhoeae. Although tagging per se appears to be required for gonococcal viability, tagging for proteolysis does not. This suggests that the essential roles of tmRNA in N.gonorrhoeae may include resolving stalled translation complexes and/or preventing depletion of free ribosomes. Although derivatives of N.gonorrhoeae expressing Escherichia coli tmRNA as their sole tmRNA were isolated, they appear to form colonies only after acquiring an extragenic suppressor(s).

Analysis of the role of trans-translation in the requirement of tmRNA for lambdaimmP22 growth in Escherichia coli.

The small, stable RNA molecule encoded by ssrA, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid lambdaimmP22 phages in Escherichia coli. tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins. A model describing this function of tmRNA, the trans-translation model (K. C. Keiler, P. R. Waller, and R. T. Sauer, Science 271:990-993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA. Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity. However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific. In light of this new information, we examine the effects on lambdaimmP22 growth of mutations eliminating activities postulated to be important for two different steps in the trans-translation model, alanine charging of tmRNA and degradation of tagged proteins. This mutational analysis suggests that, while charging of tmRNA with alanine is essential for lambdaimmP22 growth in E. coli, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth. Based on these results, we propose that trans-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation.

Pharmacokinetics of benzo[a]pyrene in the rat.

Groups of 4 male Wistar rats were dosed intravenously with 14C-labeled benzo[a]pyrene dissolved in an Emulphor/water vehicle at 3 different dose levels and killed at 1 of 15 specific time intervals from 5 min to 32 h after dosing. 14C-Radiolabel concentration-time data were obtained for blood, brain, adipose, heart, kidney, liver, lung, spleen, and testes. Benzo[a]pyrene concentration-time data were obtained for blood, adipose, kidney, liver, and lung. Appropriate mathematical models were fitted to these data and to the data for metabolites derived as the residuals from 14C-radiolabel minus benzo[a]pyrene difference, where applicable. Nonlinear kinetics were found for 14C-radiolabel in liver, while the data from lung for both 14C-radiolabel and for benzo[a]pyrene per se supported the binding of benzo[a]pyrene in that tissue.

Physiologically-based pharmacokinetic modeling of pyrene in the rat.

The objective of the present study was to develop a physiologically-based model to simulate the oral and i.v. pharmacokinetics of pyrene in the rat. The physiologically-based pharmacokinetic (PBPK) model for pyrene consisted of the following tissue compartments: liver, lungs, adipose tissue, slowly perfused tissues, and richly perfused tissues interconnected with arterial and venous blood pools. The tissue:blood partition coefficients required for the pyrene PBPK model were estimated by equilibrium dialysis. Using perfusion-limited descriptions for tissue uptake and previously determined in vitro-derived hepatic metabolism rate constants (V(max) and K(m)), the PBPK model predicted a faster clearance of pyrene than that suggested by the experimental data. The biological basis of PBPK model then provided an opportunity to refine the estimate of V(max), and to explore and uncover additional mechanistic determinants of pyrene disposition in vivo. Accordingly, the in vitro V(max) had to be lowered by about a factor of 10 to adequately simulate experimental data on pyrene pharmacokinetics. Further, the model simulations could be matched with the experimental data on tissue concentrations of pyrene only with the considerations of (i) diffusion-limited uptake in slowly perfused tissues and adipose tissue, and (ii) binding to proteins in metabolizing tissues (lungs and liver). The present study successfully integrated the available data on oral and i.v. pharmacokinetics of pyrene using a physiological model framework, and identified several mechanistic data gaps that should be addressed by future research efforts.

Rate of pyrene metabolism in rat liver post-mitochondrial fractions.

The objective of the present study was to estimate the maximal velocity (Vmax) and Michaelis affinity constant (Km) for the oxidation of pyrene to 1-hydroxypyrene using rat liver post-mitochondrial fractions. The approach involved the determination of the concentrations of 1-hydroxypyrene formed during 5 min incubations of pyrene (initial concentrations: 0.0025-0.5 microM), and correcting for the rate of 1-hydroxypyrene disappearance (2.16 x 10(-5) per (mg protein/l)/min) during the incubation period. The Vmax and Km for pyrene metabolism in the rat corresponded to 0.0577 +/- 0.0108 micromol/min per g liver and 27.73 +/- 13.54 microM, respectively. The intrinsic clearance (CL(int)) of pyrene in the rat estimated in the present study (0.041-0.111 l/min per kg) was within the range of the previously reported CL(int) in humans (0.037-0.125 l/min per kg). The results of this study suggest that CL(int) of pyrene in humans can be predicted from such data obtained in the rat.

Methods of assessing dermal absorption with emphasis on uptake from contaminated vegetation.

Dermal absorption may be an important route of exposure in several exposure scenarios for workers and the general public. Because criteria (e.g. RfDs or MRLs) for chemical exposures are usually expressed in units of mg/kg/day, risk assessments often attempt to convert dermal exposure data to units of mg/kg/day absorbed dose. For some types of dermal exposure involving direct and continuous contact with liquids, Fick's first law may be used. In other cases, such as those involving spills onto the skin surface or dermal exposure to contaminated vegetation, the applicability of Fick's first law is limited. This analysis focuses on a method for estimating absorbed dose from dermal contact with contaminated vegetation or other surfaces. The method involves two steps: estimating the transfer rate from contaminated vegetation to the skin surface, and estimating the extent of absorption from the skin surface into the body. A generic equation can be derived for estimating the transfer rate (TR) from dislodgeable foliar residues (DFR): logTR = 1.09 logDFR + 0.05. Given the surface area of the exposed skin and the duration of contact, this equation can be used to estimate the amount of chemical deposited on the surface of the skin. This equation is based on data from eight different studies using 16 different pesticides. Excluding one outlier (Vinclozolin), the squared correlation coefficient for this equation is 0.78, and the model is significant at p < 0.00001. Data from a series of studies by Feldmann and Maibach (1969, 1970, 1974) are used to estimate dermal absorption. These studies were selected as the most relevant for risk assessment because most of the experimental subjects are human and because of the nature of dermal exposures was closely related to many exposure scenarios used in risk assessments. For all 47 compounds included in this series of studies, there were no significant correlations between commonly available physical or chemical properties and dermal absorption. For those compounds with a K0/w > 1.85, however, the average daily absorption rate (AR) over a five-day postexposure period can be estimated from the molecular weight: logAR = -0.04MW + 1.5. The squared correlation coefficient for this equation is 0.68, and the model is significant at p < 0.00001. The usefulness of this approach is evaluated using a study by Harris and Solomon (1992) in which the absorption of 2,4-D from contaminated turf was measured in a group of volunteers. The estimated absorbed dose using the equations above is very close to the measured values.

Applications of physiologic pharmacokinetic modeling in carcinogenic risk assessment.

The use of physiologically based pharmacokinetic (PBPK) models has been proposed as a means of estimating the dose of the reactive metabolites of carcinogenic xenobiotics reaching target tissues, thereby affording an opportunity to base estimates of potential cancer risk on tissue dose rather than external levels of exposure. In this article, we demonstrate how a PBPK model can be constructed by specifying mass-balance equations for each physiological compartment included in the model. In general, this leads to a system of nonlinear partial differential equations with which to characterize the compartment system. These equations then can be solved numerically to determine the concentration of metabolites in each compartment as functions of time. In the special case of a linear pharmacokinetic system, we present simple closed-form expressions for the area under the concentration-time curves (AUC) in individual tissue compartments. A general relationship between the AUC in blood and other tissue compartments is also established. These results are of use in identifying those parameters in the models that characterize the integrated tissue dose, and which should therefore be the primary focus of sensitivity analyses. Applications of PBPK modeling for purposes of tissue dosimetry are reviewed, including models developed for methylene chloride, ethylene oxide, 1,4-dioxane, 1-nitropyrene, as well as polychlorinated biphenyls, dioxins, and furans. Special considerations in PBPK modeling related to aging, topical absorption, pregnancy, and mixed exposures are discussed. The linkage between pharmacokinetic models used for tissue dosimetry and pharmacodynamic models for neoplastic transformation of stem cells in the target tissue is explored.

Pharmacokinetics of inhaled pyrene in rats.

Male Wistar rats were exposed to micronized aerosol concentrations of a 14C-labeled model polycyclic aromatic hydrocarbon (pyrene) at 200, 500, and 800 mg/m3 for a period of 95 min. Both the 14C label and free pyrene were monitored in the blood, urine, and feces. At the termination of the blood sampling, three of the six rats per dose group were killed and the distribution of [14C]pyrene to eight major tissues was analyzed. The analysis of blood concentration data using a one-compartment pharmacokinetic model revealed that the uptake and elimination kinetic parameters were dose dependent, for both total radioactivity (pyrene plus metabolites) and for pyrene per se, over the range of exposures used in this study. The ratio of the percent excreted via the urinary and fecal routes, collected over a 5-d period postexposure was about 1.0 at each exposure level.

Percutaneous uptake, distribution, and excretion of pyrene in rats.

Groups of 12 male Wistar rats, of about 400 g body weight, were dosed with 2, 6, or 15 mg/kg of 14C-labeled pyrene, dissolved in acetone, applied to 4 cm2 of a shaved area of the mid back. Three animals in each dose group were killed at 1, 2, 4, and 6 d post-dosing, and their principal organs were removed and analyzed for pyrene and [14C]pyrene equivalents. Urine and feces, as well as the area of skin to which the dose was applied, were also analyzed for [14C]pyrene equivalents. The rate of uptake from the skin was rapid (t1/2 0.5-0.8 d) relative to rate processes for the other organs, and about 50% of the applied dose was excreted over the 6 d of the study. The significant decrease in the fraction of the dose excreted and in the normalized amounts distributed to the various organs and tissues, as the dose increased for all chemical species measured, was strongly suggestive of nonlinear kinetics, as has been observed in previous studies. Levels of pyrene were highest in the liver, kidneys, and fat. Levels of metabolites were also high in the lung. It was evident that the dermal route of uptake was not insignificant for this model polycyclic aromatic hydrocarbon and may represent a significant exposure route for exposed humans.

Distribution of benzo[a]pyrene in pregnant rats following inhalation exposure and a comparison with similar data obtained with pyrene.

Eight pregnant rats were exposed, on the 17th day of gestation, for 95 min to a microcondensation aerosol of benzo[a]pyrene at five different atmospheric concentrations between 200 and 800 mg m-3 in a 'head-only' inhalation chamber. Five rats were killed immediately following the exposure and three were killed at 6 h post-dosing. Concentrations of the radiolabel and 'free' benzo[a]pyrene were measured in the individual fetuses and in the maternal blood, fat, kidney, liver and lung. Distribution to the fetus did not appear to be related to its position on the uterine horn and the uptake of benzo[a]pyrene was non-linear with increasing exposure concentrations, which was similar to the observations previously reported for pyrene. The levels of benzo[a]pyrene were much higher in the fetus and, especially, the lung than those observed in the pyrene study; so also were the levels of total metabolites in these tissues, which might, in part, account for the carcinogenic potency of benzo[a] pyrene.

Physiological pharmacokinetics and cancer risk assessment.

There has been considerable progress in recent years in developing physiological models for the pharmacokinetics of toxic chemicals and in the application of these models in cancer risk assessment. Physiological pharmacokinetic models consist of a number of individual compartments, based on the anatomy and physiology of the mammalian organism of interest, and include specific parameters for metabolism, tissue binding, and tissue reactivity. Because of the correspondence between these compartments and specific tissues or groups of tissues, these models are particularly useful for predicting the doses of biologically active forms of toxic chemicals at target tissues under a wide variety of exposure conditions and in different animal species, including humans. Due to their explicit characterization of the biological processes governing pharmacokinetic behaviour, these models permit more accurate predictions of the dose of active metabolites reaching target tissues in exposed humans and hence of potential cancer risk. In addition, physiological models also permit a more direct evaluation of the impact of parameter uncertainty and inter-individual variability in cancer risk assessment. In this article, we review recent developments in physiologic pharmacokinetic modeling for selected chemicals and the application of these models in carcinogenic risk assessment. We examine the use of these models in integrating diverse information on pharmacokinetics and pharmacodynamics and discuss challenges in extending these pharmacokinetic models to reflect more accurately the biological events involved in the induction of cancer by different chemicals.

Distribution to the fetus and major organs of the rat following inhalation exposure to pyrene.

Concentrations of pyrene and total metabolites were determined for individual fetuses and selected maternal organs and tissues immediately and 6 h following a 95-min head-only exposure of pregnant Wistar rats, on gestation day 17, to five levels of pyrene over the range 200-800 mg m-3 as a microcondensation aerosol. The influence of uterine horn, side and position, on distribution to the fetus was assessed. The concentration of both pyrene and its metabolites increased more over the exposure range (eightfold) than did those in the fetus. Concentrations of pyrene or its metabolites in fetal tissues were not found to be related to its position on the uterine horn. There was an unexplained and significant (P less than 0.01) higher concentration of pyrene in fetuses on the right side relative to the left side of the uterine horn for the animals killed immediately following exposure. A comparison of the levels in maternal tissues measured immediately following the exposure and 6 h later showed that there was some redistribution of pyrene and its metabolites to the fat tissues, i.e. levels in the fat increased over the 6 h period following the exposure. Levels in the other tissues diminished during this period. In general, concentrations of pyrene and its metabolites were lowest in the fetal tissues relative to those in the sampled maternal organs and tissues.

Pharmacokinetics and bioavailability of pyrene in the rat.

Groups of 6 male Wistar rats, of about 400 g body weight, were dosed with 14C-labeled pyrene, dissolved in an Emulphor/water solvent vehicle, at 5 different dose levels by the intravenous or oral routes. Appropriate mathematical models were fitted to blood concentration-time data for [14C]pyrene and pyrene per se and dose-trend analyses were carried out. Areas under these curves were used to assess the bioavailability of the orally administered doses. Tissue concentrations, measured at the termination of the blood sampling period, gave a quantitative measure of the distribution of the administered dose. Attempts to repeat these studies with similar doses of tritium-labeled benzo[a]pyrene were frustrated by the lack of meaningful blood-level data. Dose trends for the derived pharmacokinetic parameters for pyrene revealed that the kinetics were nonlinear and strongly suggestive of enterohepatic recycling. Biliary excretion, measured in a separate experiment, gave support to this hypothesis. The bioavailability of the orally administered doses was between 50 and 60%. Over a 6-d period postdosing, some 45 and 40% of the administered dose was excreted via the urine and feces, respectively, irrespective of the route of administration. Distribution to the tissues of the 14C-label was highest in the perirenal fat, intermediate in the liver, kidneys, and lungs, and lowest in the heart, testes, spleen, and brain.

Carcinogenic risk assessment of complex mixtures.

Although procedures for assessing the carcinogenic risks associated with exposure to individual chemicals are relatively well developed, risk assessment methods for mixtures of chemicals are still in the developmental stage. In this paper, we examine the difficulties in assessing the risks of exposure to complex mixtures, with special reference to the potential for synergistic effects among the components of the mixture. Statistical models for describing the joint action of multiple exposures are reviewed, and their implications for low-dose risk assessment are examined. The potential use of pharmacokinetic models to describe the metabolism of mixtures is also considered. Application of these results in regulating mixtures of carcinogenic substances is illustrated using examples involving multiple contaminants in drinking water and polycyclic aromatic hydrocarbons produced from combustion sources.

Recent developments in carcinogenic risk assessment.

In this paper, recent developments in the quantitative assessment of carcinogenic risks based on toxicological and epidemiological data are reviewed. In particular, model-free approaches to low-dose risk assessment which involve only the assumption of low-dose linearity are considered. Measures of carcinogenic potency which avoid the need to extrapolate to low doses are also described. The allometric bases for converting risk estimates between species are then discussed. Pharmacokinetic models for determining the dose delivered to the target tissue are examined, and the implications of using such models in extrapolating between doses, of exposure, and species are examined. The application of these concepts in chemical and radiation carcinogenesis is illustrated by means of brief case studies of methylene chloride and Rn. Biologically motivated cancer models based on the initiation-promotion-progression theory of carcinogenesis are discussed and compared with the classical multistage model. The estimation of risks with time-dependent exposure patterns is considered, and conditions under which the use of a time-weighted average dose is appropriate are identified. Finally, the estimation of carcinogenic risks posed by exposure to complex mixtures is explored.

Methods for route-to-route extrapolation of dose.

Results of acute toxicity studies for a variety of chemicals have indicated that, in most cases, although the inhalation route was more effective than the IG route, wide variations in toxicity occurred between these two routes. The major factors that may result in variations in toxicity between routes include: differences in absorption efficiency; differences in systemic effects; occurrence of critical toxicological effects at the portal of entry; first-pass effects resulting in inactivation or activation of the chemical agent before it reaches the target organ; and variations in temporal patterns of target organ concentrations. Extrapolation to determine safe exposure levels during chronic exposure becomes less reliable, not only as information relating to these factors decreases, but also as the quality or length of exposure decreases in the available toxicologic studies. VDC is an example of one of a few chemicals for which both chronic inhalation and oral toxicity data are available, along with detailed pharmacokinetic information. On the basis of the pharmacokinetic data, differences in toxicity between the two routes did not appear to be very likely for this chemical. This conjecture was supported by the results of chronic toxicity studies. Finally, assuming sufficient data and pharmacokinetic parameters are available, this paper presents a useful and practical approach to route extrapolation.