Use of in vitro data in developing a physiologically based pharmacokinetic model: Carbaryl as a case study.

In vitro-derived information has been increasingly used to support and improve human health risk assessment for exposure to chemicals. Physiologically based pharmacokinetic (PBPK) modeling is a key component in the movement toward in vitro-based risk assessment, providing a tool to integrate diverse experimental data and mechanistic information to relate in vitro effective concentrations to equivalent human exposures. One of the challenges, however, in the use of PBPK models for this purpose has been the need for extensive chemical-specific parameters. With the remarkable advances in in vitro methodologies in recent years, in vitro-derived parameters can now be easily incorporated into PBPK models. In this study we demonstrate an in vitro data based parameterization approach to develop a physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model, using carbaryl as a case study. In vitro experiments were performed to provide the chemical-specific pharmacokinetic (PK) and pharmacodynamic (PD) parameters for carbaryl in the PBPK model for this compound. Metabolic clearance and cholinesterase (ChE) interaction parameters for carbaryl were measured in rat and human tissues. These in vitro PK and PD data were extrapolated to parameters in the whole body PBPK model using biologically appropriate scaling. The PBPK model was then used to predict the kinetics and ChE inhibition dynamics of carbaryl in vivo. This case study with carbaryl provides a reasonably successful example of utilizing the in vitro to in vivo extrapolation (IVIVE) approach for PBPK model development. This approach can be applied to other carbamates with an anticholinesterase mode of action as well as to environmental chemicals in general with further refinement of the current shortcomings in the approach. It will contribute to minimizing the need for in vivo human data for PBPK model parameterization and evaluation in human risk assessments.

Cytotoxicity of naphthalene toward cells from target and non-target organs in vitro.

Chronic inhalation exposure to high concentrations of naphthalene produced nasal tumors in rats and lung tumors in female mice. Naphthalene bioactivation is required for target organ toxicity and cytotoxicity in target organs may be involved in tumor development. The present studies characterized the dose-response relationships for naphthalene-induced glutathione (GSH) depletion, effects on cellular ATP, and cytotoxicity in cells from both target (lung, nasal epithelium) and non-target (liver) organs in vitro using cells from F-344 rats, B6C3F1 mice and humans. The cells were incubated with various concentrations of naphthalene in sealed glass flasks for 3h, then placed in monolayer culture in fresh media for 24h to examine the repair or progression of damage. Naphthalene was a low potency cytotoxicant in vitro, with 500 µM frequently observed as a no-observed adverse effect concentration or lowest observed adverse effect concentration. Naphthalene exposure produced dose-dependent decreases in cellular GSH, ATP and viability in rat, mouse and human hepatocytes at concentrations >500 µM. Human nasal respiratory epithelial cells exhibited greater naphthalene cytotoxicity than rat or mouse nasal respiratory epithelial cell preparations. Rat nasal respiratory epithelial cell preparations metabolized naphthalene through pathways leading to the preferential formation of 1,2-naphthoquinone GSH conjugates rather than 1,4-naphthoquinone GSH conjugates observed in rat hepatocytes or mouse nasal respiratory epithelial cells, consistent with the suggestion that this bioactivation pathway may be involved in rat nasal tumor development. Naphthalene exposures of ≥500 µM decreased cellular GSH and ATP in rat, mouse and human lung cell preparations. The variability of the responses of the human lung cell preparations was consistent with the known variability of CYP activities in human lung tissue. The results of these studies can be used as the basis for future studies of the mechanisms involved in naphthalene-induced cytotoxicity and the relevance of the bioactivation pathways for human exposure to naphthalene.

Application of an updated physiologically based pharmacokinetic model for chloroform to evaluate CYP2E1-mediated renal toxicity in rats and mice.

Physiologically based pharmacokinetic (PBPK) models are tools for interpreting toxicological data and extrapolating observations across species and route of exposure. Chloroform (CHCl(3)) is a chemical for which there are PBPK models available in different species and multiple sites of toxicity. Because chloroform induces toxic effects in the liver and kidneys via production of reactive metabolites, proper characterization of metabolism in these tissues is essential for risk assessment. Although hepatic metabolism of chloroform is adequately described by these models, there is higher uncertainty for renal metabolism due to a lack of species-specific data and direct measurements of renal metabolism. Furthermore, models typically fail to account for regional differences in metabolic capacity within the kidney. Mischaracterization of renal metabolism may have a negligible effect on systemic chloroform levels, but it is anticipated to have a significant impact on the estimated site-specific production of reactive metabolites. In this article, rate parameters for chloroform metabolism in the kidney are revised for rats, mice, and humans. New in vitro data were collected in mice and humans for this purpose and are presented here. The revised PBPK model is used to interpret data of chloroform-induced kidney toxicity in rats and mice exposed via inhalation and drinking water. Benchmark dose (BMD) modeling is used to characterize the dose-response relationship of kidney toxicity markers as a function of PBPK-derived internal kidney dose. Applying the PBPK model, it was also possible to characterize the dose response for a recent data set of rats exposed via multiple routes simultaneously. Consistent BMD modeling results were observed regardless of species or route of exposure.

Dose-response assessment of naphthalene-induced genotoxicity and glutathione detoxication in human TK6 lymphoblasts.

The dose-response relationship for the induction of micronuclei (MN) and the impact of glutathione (GSH) detoxication on naphthalene-induced cytotoxicity and genotoxicity were investigated in human TK6 cells. TK6 cells were exposed to 10 concentrations ranging from 0.0625 to 30µM naphthalene in the presence of ß-naphthoflavone- and phenobarbital (ßNP/PB)-induced rat liver S9 with a nicotinamide adenine dinucleotide phosphate-generating system. Three approaches were used to identify a no-observed-effect level (NOEL) for naphthalene-induced genotoxicity: (1) laboratory criteria of ≥ twofold increase over the concurrent solvent controls (NOEL = 10µM), (2) ANOVA with Bonferroni correction (NOEL = 2.5µM), and (3) the benchmark dose approach (BMCL(10) = 3.35µM). The NOEL and point of departure micronucleus frequency for naphthalene-induced MN are between the tested naphthalene concentrations of 2.5-10.0µM in this experimental system. Supplementation of the exposure system with physiological relevant concentrations of 5mM GSH eliminated naphthalene-induced cytotoxicity and genotoxicity; no increased cytotoxicity or genotoxicity was observed at concentrations of up to 500µM naphthalene in the presence of GSH compared with 2.5-10.0µM in the absence of GSH. Naphthalene bioactivation by ßNP/PB-induced rat liver S9 exhibits a nonlinear dose-response for the induction of MN in TK6 cells with a NOEL of 2.5-10µM that in the presence of GSH is shifted upward greater than 50- to 200-fold. These data demonstrate a nonlinear dose-response for naphthalene-induced genotoxicity that is eliminated by GSH, and both observations should be considered when assessing human risk from naphthalene exposures.

Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes.

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic(III) is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic(III) can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenic(III) (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA(III) from sodium arsenite (1 microM) was linear with respect to time for >90 min. DMA(III) formation did not become significant until 60 min. MMA(V) and DMA(V) were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic(V) was not observed in mouse hepatocytes, consistent with inhibition of arsenic(V) active cellular uptake by phosphate in the medium. The formation of MMA(III) increased with increasing arsenic(III) concentrations up to approximately 2 microM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic(III). Kinetic analysis of the data suggested an apparent K(M) of approximately 3.6 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM arsenic(III). The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA(III) in liver and other tissues.

Incorporation of pharmacokinetic and pharmacodynamic data into risk assessments.

Risk assessment methodologies are being updated to allow the inclusion of numerical values for variance in pharmacokinetic (PK) measures and pharmacodynamic (PD) processes related to toxicity. The key PK measures and PD processes are identified from the results of carefully conducted and adequately reported studies. In some instances, studies with humans are not possible, and so the development of data useful for human PK evaluations and on PD processes in vitro or in silico represent an alternative. These results can be integrated under physiologic, anatomic, and biochemical constraints of the intact body through physiologically based pharmacokinetic (PBPK) modeling. This manuscript presents the rational for and key considerations related to the inclusion of quantitative PK and PD data in assessing chemical risks.

The impact of cytochrome P450 2E1-dependent metabolic variance on a risk-relevant pharmacokinetic outcome in humans.

Risk assessments include assumptions about sensitive subpopulations, such as the fraction of the general population that is sensitive and the extent that biochemical or physiological attributes influence sensitivity. Uncertainty factors (UF) account for both pharmacokinetic (PK) and pharmacodynamic (PD) components, allowing the inclusion of risk-relevant information to replace default assumptions about PK and PD variance (uncertainty). Large numbers of human organ donor samples and recent advances in methods to extrapolate in vitro enzyme expression and activity data to the intact human enable the investigation of the impact of PK variability on human susceptibility. The hepatotoxicity of trichloroethylene (TCE) is mediated by acid metabolites formed by cytochrome P450 2E1 (CYP2E1) oxidation, and differences in the CYP2E1 expression are hypothesized to affect susceptibility to TCE's liver injury. This study was designed specifically to examine the contribution of statistically quantified variance in enzyme content and activity on the risk of hepatotoxic injury among adult humans. We combined data sets describing (1) the microsomal protein content of human liver, (2) the CYP2E1 content of human liver microsomal protein, and (3) the in vitro Vmax for TCE oxidation by humans. The 5th and 95th percentiles of the resulting distribution (TCE oxidized per minute per gram liver) differed by approximately sixfold. These values were converted to mg TCE oxidized/h/kg body mass and incorporated in a human PBPK model. Simulations of 8-hour inhalation exposure to 50 ppm and oral exposure to 5 micro g TCE/L in 2 L drinking water showed that the amount of TCE oxidized in the liver differs by 2% or less under extreme values of CYP2E1 expression and activity (here, selected as the 5th and 95th percentiles of the resulting distribution). This indicates that differences in enzyme expression and TCE oxidation among the central 90% of the adult human population account for approximately 2% of the difference in production of the risk-relevant PK outcome for TCE-mediated liver injury. Integration of in vitro metabolism information into physiological models may reduce the uncertainties associated with risk contributions of differences in enzyme expression and the UF that represent PK variability.

Physiologically based pharmacokinetic model parameter estimation and sensitivity and variability analyses for acrylonitrile disposition in humans.

A physiologically based pharmacokinetic (PBPK) model of acrylonitrile (ACN) and cyanoethylene oxide (CEO) disposition in humans was developed and is based on human in vitro data and scaling from a rat model (G. L. Kedderis et al., 1996, TOXICOL: Appl. Pharmacol.140, 422-435) for application to risk assessment. All of the major biotransformation and reactivity pathways, including metabolism of ACN to glutathione conjugates and CEO, reaction rates of ACN and CEO with glutathione and tissues, and the metabolism of CEO by hydrolysis and glutathione conjugation, were described in the human PBPK model. Model simulations indicated that predicted blood and brain ACN and CEO concentrations were similar in rats and humans exposed to ACN by inhalation. In contrast, rats consuming ACN in drinking water had higher predicted blood concentrations of ACN than humans exposed to the same concentration in water. Sensitivity and variability analyses were conducted on the model. While many parameters contributed to the estimated variability of the model predictions, the reaction rate of CEO with glutathione, hydrolysis rate for CEO, and blood:brain partition coefficient of CEO were the parameters predicted to make the greatest contributions to variability of blood and brain CEO concentrations in humans. The main contributor to predicted variance in human blood ACN concentrations in people exposed through drinking water was the Vmax for conversion of ACN to CEO. In contrast, the main contributors for variance in people exposed by inhalation were expected to be the rate of blood flow to the liver and alveolar ventilation rate, with the brain:blood partition coefficient also contributing to variability in predicted concentrations of ACN in the brain. Expected variability in blood CEO concentrations (peak or average) in humans exposed by inhalation or drinking water was modest, with a 95th-percentile individual expected to have blood concentrations 1.8-times higher than an average individual.

Priorities for development of research methods in occupational cancer.

Occupational cancer research methods was identified in 1996 as 1 of 21 priority research areas in the National Occupational Research Agenda (NORA). To implement NORA, teams of experts from various sectors were formed and given the charge to further define research needs and develop strategies to enhance or augment research in each priority area. This article is a product of that process. Focus on occupational cancer research methods is important both because occupational factors play a significant role in a number of cancers, resulting in significant morbidity and mortality, and also because occupational cohorts (because of higher exposure levels) often provide unique opportunities to evaluate health effects of environmental toxicants and understand the carcinogenic process in humans. Despite an explosion of new methods for cancer research in general, these have not been widely applied to occupational cancer research. In this article we identify needs and gaps in occupational cancer research methods in four broad areas: identification of occupational carcinogens, design of epidemiologic studies, risk assessment, and primary and secondary prevention. Progress in occupational cancer will require interdisciplinary research involving epidemiologists, industrial hygienists, toxicologists, and molecular biologists.

Incorporating human interindividual biotransformation variance in health risk assessment.

The protection of sensitive individuals within a population dictates that measures other than central tendencies be employed to estimate risk. The refinement of human health risk assessments for chemicals metabolized by the liver to reflect data on human variability can be accomplished through (1) the characterization of enzyme expression in large banks of human liver samples, (2) the employment of appropriate techniques for the quantification and extrapolation of metabolic rates derived in vitro, and (3) the judicious application of physiologically based pharmacokinetic (PBPK) modeling. While in vitro measurements of specific biochemical reactions from multiple human samples can yield qualitatively valuable data on human variance, such measures must be put into the perspective of the intact human to yield the most valuable predictions of metabolic differences among humans. For quantitative metabolism data to be the most valuable in risk assessment, they must be tied to human anatomy and physiology, and the impact of their variance evaluated under real exposure scenarios. For chemicals metabolized in the liver, the concentration of parent chemical in the liver represents the substrate concentration in the Michaelis Menten description of metabolism. Metabolic constants derived in vitro may be extrapolated to the intact liver, when appropriate conditions are met. Metabolic capacity Vmax; the maximal rate of the reaction) can be scaled directly to the concentration of enzyme (or enzyme fraction) contained in the liver. Several environmental, genetic and lifestyle factors can influence the concentration of cytochrome P450 forms (CYP) in the liver by affecting either (1) the extent to which the CYP forms are expressed in the endoplasmic reticulum of the cell (isolated as the microsomal fraction from tissue homogenates), or (2) the expression of microsomal protein in intact liver tissue. Biochemically sound measures of the hepatic distribution of xenobiotic metabolizing enzymes among humans, based on expression of the enzymes within microsomal protein and the distribution of microsomal protein among intact livers, can be combined with metabolic constants derived in vitro to generate values consistent with those employed in PBPK models. When completed, the distribution (and bounds) of Vmax values can be estimated and included in PBPK models. Exercising such models under plausible exposure scenarios will demonstrate the extent to which human interindividual enzyme variance can influence parameters (i.e., the detoxication of a toxic chemical through metabolism) that may influence risk. In this article, we describe a methodology and conditions which must exist for such an approach to be successful.