Global optimization of the Michaelis-Menten parameters using physiologically-based pharmacokinetic (PBPK) modeling and chloroform vapor uptake data in F344 rats.

Objective: To quantify metabolism, a physiologically based pharmacokinetic (PBPK) model for a volatile compound can be calibrated with the closed chamber (i.e. vapor uptake) inhalation data. Here, we introduce global optimization as a novel component of the predictive process and use it to illustrate a procedure for metabolic parameter estimation.Materials and methods: Male F344 rats were exposed in vapor uptake chambers to initial concentrations of 100, 500, 1000, and 3000 ppm chloroform. Chamber time-course data from these experiments, in combination with optimization using a chemical-specific PBPK model, were used to estimate Michaelis-Menten metabolic constants. Matlab® simulation software was used to integrate the mass balance equations and to perform the global optimizations using MEIGO (MEtaheuristics for systems biology and bIoinformatics Global Optimization - Version 64 bit, R2016A), a toolbox written for Matlab®. The cost function used the chamber time-course data and least squares to minimize the difference between data and simulation values.Results and discussion: The final values estimated for Vmax (maximum metabolic rate) and Km (affinity constant) were 1.2 mg/h and a range between 0.0005 and 0.6 mg/L, respectively. Also, cost function plots were used to analyze the dose-dependent capacity to estimate Vmax and Km within the experimental range used. Sensitivity analysis was used to assess identifiability for both parameters and show these kinetic data may not be sufficient to identify Km.Conclusion: In summary, this work should help toxicologists interested in optimization techniques understand the overall process employed when calibrating metabolic parameters in a PBPK model with inhalation data.

Chlorination of Source Water Containing Iodinated X-ray Contrast Media: Mutagenicity and Identification of New Iodinated Disinfection Byproducts.

Iodinated contrast media (ICM) are nonmutagenic agents administered for X-ray imaging of soft tissues. ICM can reach µg/L levels in surface waters because they are administered in high doses, excreted largely unmetabolized, and poorly removed by wastewater treatment. Iodinated disinfection byproducts (I-DBPs) are highly genotoxic and have been reported in disinfected waters containing ICM. We assessed the mutagenicity in Salmonella of extracts of chlorinated source water containing one of four ICM (iopamidol, iopromide, iohexol, and diatrizoate). We quantified 21 regulated and nonregulated DBPs and 11 target I-DBPs and conducted a nontarget, comprehensive broad-screen identification of I-DBPs. We detected one new iodomethane (trichloroiodomethane), three new iodoacids (dichloroiodoacetic acid, chlorodiiodoacetic acid, bromochloroiodoacetic acid), and two new nitrogenous I-DBPs (iodoacetonitrile and chloroiodoacetonitrile). Their formation depended on the presence of iopamidol as the iodine source; identities were confirmed with authentic standards when available. This is the first identification in simulated drinking water of chloroiodoacetonitrile and iodoacetonitrile, the latter of which is highly cytotoxic and genotoxic in mammalian cells. Iopamidol (5 µM) altered the concentrations and relative distribution of several DBP classes, increasing total haloacetonitriles by >10-fold. Chlorination of ICM-containing source water increased I-DBP concentrations but not mutagenicity, indicating that such I-DBPs were either not mutagenic or at concentrations too low to affect mutagenicity.

In vitro metabolism of thyroxine by rat and human hepatocytes.

1. The liver metabolizes thyroxine (T(4)) through two major pathways: deiodination and conjugation. Following exposure to xenobiotics, T(4) conjugation increases through the induction of hepatic uridine diphosphate glucuronosyltransferase (UGT) in rodents; however, it is uncertain to what degree different species employ deiodination and conjugation in the metabolism of T(4). The objective of this study was to compare the metabolism of T4 in untreated and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-treated primary sandwich-cultured hepatocytes from rat (SCRH) and human (SCHH). 2. Basal metabolite concentrations were 13 times higher in the medium of SCRH compared to SCHH. Metabolite distribution in the medium of SCRH versus SCHH was as follows: T(4)G, (91.6 versus 5.3%); T4S, (3.6 versus 4.4%) and T(3) + rT(3), (4.9 versus 90.3%). PCB 153 induced T(4)G in the medium of SCRH and SCHH; however, T(4)S and T(3) + rT(3) were changed but to a much lesser degree. 3. The results indicate that baseline T(4) glucuronidation is greater in SCRH compared to SCHH. These data also suggest that glucuronidation may be a more important pathway for T(4) metabolism in rats and deiodination may be a favored pathway in humans; however, with PCB 153 treatment these data support glucuronidation as a primary route of T(4) metabolism in both rat and humans.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

Integrated disinfection by-products mixtures research: assessment of developmental toxicity in Sprague-Dawley rats exposed to concentrates of water disinfected by chlorination and ozonation/postchlorination.

Epidemiological and animal toxicity studies have raised concerns regarding possible adverse health effects of disinfection by-products (DBPs) found in drinking water. The classes and concentrations of DBPs are influenced by the choice of disinfection process (e.g., chlorination, ozonation) as well as source water characteristics (e.g., pH, total organic carbon, bromide content). Disinfected waters were found to contain more than 500 compounds, many of which remain unidentified. Therefore, a "whole-mixture" approach was used to evaluate the toxic potential of alternative disinfection scenarios. An in vivo developmental toxicity screen was used to evaluate the adverse developmental effects of the complex mixtures produced by two different disinfection processes. Water was obtained from East Fork Lake, Ohio; spiked with iodide and bromide; and disinfected either by chlorination or by ozonation/postchlorination, producing finished drinking water suitable for human consumption. These waters were concentrated approximately 130-fold by reverse osmosis membrane techniques. To the extent possible, volatile DBPs lost in the concentration process were spiked back into the concentrates. These concentrates were then provided as drinking water to Sprague-Dawley rats on gestation days 6-16; controls received boiled, distilled, deionized water. The dams (19-20 per group) were allowed to deliver and their litters were examined on postnatal days (PD) 1 and 6. All dams delivered normally, with parturition occurring significantly earlier in the ozonation/postchlorination group. However, no effects on prenatal survival, postnatal survival, or pup weight were evident. Skeletal examination of the PD-6 pups also revealed no treatment effects. Thus, approximately 130-fold higher concentrates of both ozonated/postchlorinated and chlorinated water appeared to exert no adverse developmental effects in this study.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα). Male B6C3F1 mice were exposed for 7 days to di (2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), and n-butyl benzyl phthalate (BBP), which vary in PPARα activity and liver tumorigenicity. Each phthalate increased expression of select PPARα target genes at 7 days, while only DEHP significantly increased liver cell proliferation labeling index (LI). Transcriptional benchmark dose (BMDT) estimates for dose-related genomic markers stratified phthalates according to hypothetical tumorigenic potencies, unlike BMDs for non-genomic endpoints (relative liver weights or proliferation). The 7-day BMDT values for Acot1 as a surrogate measure for PPARα activation were 29, 370, and 676 mg/kg/day for DEHP, DNOP, and BBP, respectively, distinguishing DEHP (liver tumor BMD of 35 mg/kg/day) from non-tumorigenic DNOP and BBP. Effect thresholds were generated using linear regression of DEHP effects at 7 days and 2-year tumor incidence values to anchor early response molecular indicators and a later phenotypic outcome. Thresholds varied widely by marker, from 2-fold (Pdk4 and proliferation LI) to 30-fold (Acot1) induction to reach hypothetical tumorigenic expression levels. These findings highlight key issues in defining thresholds for biological adversity based on molecular changes.