Mitochondrial DNA sequence and phylogenetic evaluation of geographically disparate Sus scrofa breeds.

Next generation sequencing of mitochondrial DNA (mtDNA) facilitates studies into the metabolic characteristics of production animals and their relation to production traits. Sequence analysis of mtDNA from pure-bred swine with highly disparate production characteristics (Mangalica Blonde, Mangalica Swallow-bellied, Meishan, Turopolje, and Yorkshire) was initiated to evaluate the influence of mtDNA polymorphisms on mitochondrial function. Herein, we report the complete mtDNA sequences of five Sus scrofa breeds and evaluate their position within the phylogeny of domestic swine. Phenotypic traits of Yorkshire, Mangalica Blonde, and Swallow-belly swine are presented to demonstrate their metabolic characteristics. Our data support the division of European and Asian breeds noted previously and confirm European ancestry of Mangalica and Turopolje breeds. Furthermore, mtDNA differences between breeds suggest function-altering changes in proteins involved in oxidative phosphorylation such as ATP synthase 6 (MT-ATP6), cytochrome oxidase I (MT-CO1), cytochrome oxidase III (MT-CO3), and cytochrome b (MT-CYB), supporting the hypothesis that mtDNA polymorphisms contribute to differences in metabolic traits between swine breeds. Our sequence data form the basis for future research into the roles of mtDNA in determining production traits in domestic animals. Additionally, such studies should provide insight into how mtDNA haplotype influences the extreme adiposity observed in Mangalica breeds.

Dioxin-responsive genes: examination of dose-response relationships using quantitative reverse transcriptase-polymerase chain reaction.

The purpose of the present experiments was to examine dose-response relationships for induction of hepatic mRNA following a single administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats. The induction of cytochrome P450-1A1 (CYP1A1) mRNA is compared to other "dioxin-responsive" genes including UDP-glucuronosyltransferase I, plasminogen activator inhibitor 2, and transforming growth factor alpha using a sensitive reverse transcriptase-polymerase chain reaction-based method. Sample-to-sample variability in amplification is a concern in using polymerase chain reaction to quantitate biological responses. However, in the present study recombinant RNA templates were synthesized to use as internal standards in both the reverse transcription and the polymerase chain reaction steps. The induction of CYP1A1 mRNA was extremely sensitive to TCDD treatment with increases observed at doses as low as 1 ng/kg body weight. The induction of CYP1A1 mRNA correlated highly (R2 > 0.90) with an increase in ethoxyresorufin-o-deethylase activity, a CYP1A1-associated enzyme activity. However, induction of CYP1A1 mRNA levels was detected at lower TCDD doses than was ethoxyresorufin-o-deethylase activity, reflecting the greater sensitivity of the reverse transcription-polymerase chain reaction approach to detect transcriptional activation of the CYP1A1 gene. UDP-glucuronosyltransferase I mRNA was increased over control (5-fold) but required 1000-times higher TCDD doses (1 microgram/kg) to result in a significant increase than did CYP1A1. Plasminogen activator inhibitor 2 and transforming growth factor alpha mRNA, both previously shown to be induced by TCDD in human keratinocytes, were not increased in rat liver. Hence, these studies reaffirm that TCDD acts through classical receptor mechanisms with gene-to-gene differences in responsiveness. The reverse transcription-polymerase chain reaction method developed to measure mRNA for dioxin-responsive genes in rat liver will allow for measuring multigene and tissue responses to TCDD and other xenobiotics with high sensitivity, reproducibility, and adaptability and should increase our understanding of various dose-response relationships.

Biochemical origins of the non-monotonic receptor-mediated dose-response.

A mathematical model was created to examine how xenobiotic ligands that bind to nuclear receptor proteins may affect transcriptional activation of hormone-regulated genes. The model included binding of the natural ligand (e.g. hormone) and xenobiotic ligands to the receptor, binding of the liganded receptor to receptor-specific DNA response sequences, binding of co-activator or co-repressor proteins (Rp) to the resulting complex, and the consequent transcriptional rate relative to that in the absence of the xenobiotic agent. The model predicted that the xenobiotic could act as a pure agonist, a pure antagonist, or a mixed agonist whose dose-response curve exhibits a local maximum. The response to the agent depends on the affinity of the liganded receptor-DNA complex for binding additional transcription factors (e.g. co-activator proteins). An inverted U-shaped dose-response occurred when basal levels of the natural ligand did not saturate receptor binding sites and the affinity for co-activator is weaker when the xenobiotic ligand is bound to the receptor than when the endogenous ligand is bound. The dose-response curve shape was not dependent on the affinity of the receptor for the xenobiotic agent; alteration of this value merely shifted the curve along the concentration axis. The amount of receptor, the density of DNA response sequences, and the affinity of the DNA-bound receptor for Rp determine the amplitude of the computed response with little overall change in curve shape. This model indicates that a non-monotonic dose-response is a plausible outcome for xenobiotic agents that activate nuclear receptors in the same manner as natural ligands.

The privileged access model of 1,3-butadiene disposition.

In previous attempts to model disposition of 1,3-butadiene in mice and rats, parameter values for 1,2-epoxybut-3-ene metabolism were optimized to reproduce elimination of this gas from closed chambers. However, each of these models predicted much higher concentrations of circulating epoxybutene than were subsequently measured in animals exposed to butadiene. To account for this discrepancy, a previous physiologically based pharmacokinetic model of butadiene disposition was modified to describe a transient complex between cytochrome P450 and epoxide hydrolase on the endoplasmic reticulum membrane. In this model the epoxide products are directly transferred from the P450 to the epoxide hydrolase in competition with release of products into the cytosol. The model includes flow-restricted delivery of butadiene and epoxides to gastrointestinal tract, liver, lung, kidney, fat, other rapidly perfused tissues, and other slowly perfused tissues. Blood was distributed among compartments for arterial, venous, and capillary spaces. Oxidation of butadiene and epoxybutene and hydrolysis and glutathione conjugation of epoxides were included in liver, lung, and kidney. The model reproduces observed uptake of butadiene and epoxybutene from closed chambers by mice and rats and steady-state concentrations of butadiene, epoxybutene, and 1,2;3,4-diepoxybutane concentrations in blood of mice and rats exposed by nose only. Successful replication of these observations indicates that the proposed privileged access of epoxides formed in situ to epoxide hydrolase is a plausible mechanistic representation for the metabolic clearance of epoxide-forming chemicals.

Implications for risk assessment of suggested nongenotoxic mechanisms of chemical carcinogenesis.

Nongenotoxic carcinogens are chemicals that induce neoplasia without it or its metabolites reacting directly with DNA. Chemicals classified as nongenotoxic carcinogens have been assumed to act as tumor promoters and exhibit threshold tumor dose-responses. This is in contrast to genotoxic carcinogens that are DNA reactive, act as tumor initiators, and are assumed to exhibit proportional responses at low doses. In this perspective, we examine the basic tenets and utility of this classification for evaluating human cancer risk. Two classes of so-called nongenotoxic chemical carcinogens selected for review include cytotoxic agents that induce regenerative hyperplasia (trihalomethanes and inducers of alpha 2-microglobulin nephropathy) and agents that act via receptor-mediated mechanisms (peroxisome proliferators and dioxin). Major conclusions of this review include: a) many chemicals considered to be nongenotoxic carcinogens actually possess certain genotoxic activities, and limiting evaluations of carcinogenicity to their nongenotoxic effects can be misleading; b) some nongenotoxic activities may cause oxidative DNA damage and thereby initiate carcinogenesis; c) although cell replication is involved in tumor development, cytotoxicity and mitogenesis do not reliably predict carcinogenesis; d) a threshold tumor response is not an inevitable result of a receptor-mediated mechanism. There are insufficient data on the chemicals reviewed here to justify treating their carcinogenic effects in animals as irrelevant for evaluating human risk. Research findings that characterize the multiple mechanisms of chemical carcinogenesis should be used quantitatively to clarify human dose-response relationships, leading to improved scientifically based public health decisions. Excessive reliance on oversimplified classification schemes that do not consider all potential contributing effects of a toxicant can obscure the actual causal relationships between exposure and cancer outcome.

Biochemical mechanisms and cancer risk assessment models for dioxin.

Biologically realistic mechanistic models of carcinogenesis by TCDD are composed of equations representing biochemical events leading to altered expression of proteins involved in the response or equations representing the kinetics of proliferation of clones of mutant cells. A biochemically augmented physiological dosimetry model reproduces the observed altered expression of liver proteins in female rats exposed to dioxin. The model suggests that oxidation of estradiol to DNA reactive quinones or semiquinones by CYP1A2 protein induced by TCDD may contribute to an increased mutational rate. It suggests that TCDD-stimulated production of a peptide ligand of the epidermal growth factor (EGF) receptor and subsequent activation of the receptor's tyrosine kinase activity may increase the rate of proliferation of susceptible cells. These calculated quantities can serve as indices of toxicity and can be used to predict tumor incidence as a function of exposure.

Effects of the structure of a toxicokinetic model of butadiene inhalation exposure on computed production of carcinogenic intermediates.

A flow-limited physiologically based toxicokinetic model was constructed for uptake, metabolism, and clearance of butadiene (BD) and its principal metabolite 1,2-epoxy-3-butene (EB), using physiological and biochemical parameters from the literature where available. The model includes compartments for blood, liver, lung, fat, GI tract, other rapidly perfused tissues, and slowly perfused tissues. The blood was distributed among compartments for arterial plus venous blood and subcompartments for vascular spaces associated with each of the tissue compartments. The lung contained a subcompartment for the alveolar space. Metabolic activation of BD by cytochrome P450-catalyzed epoxidation was modeled as occurring in liver, lung, and the rapidly perfused tissue compartments. The detoxication of EB catalyzed by epoxide hydrolase and glutathione S-transferase (GST) was modeled as occurring in liver, lung, and the rapidly perfused tissues compartments and by blood GST activity. The model also includes depletion of glutathione (GSH) by GST-catalyzed conjugation of EB and 3-butene-1,2-diol and resynthesis of GSH from cysteine. Values of biochemical parameters that were unavailable in the literature were estimated by iteratively reweighted least squares optimization to reproduce data for uptake of BD and EB by rats and mice in closed chambers. The resulting model also reproduced the depletion of GSH in liver and lung in flow-through systems. It reproduced the concentrations of expired EB produced from BD in closed chambers but overpredicted separately measured blood EB concentrations in flow-through systems, indicating an inconsistency between these two experiments that cannot be resolved by this model or an inadequacy in the model. Equilibration of chamber gases with the alveolar space and alveolar gas with lung capillary blood results in much less dilution of the inhaled gas in the blood compared with the predictions of models in which chamber gas equilibrates directly with the total circulation. The production of EB predicted by the present model was found to be sensitive to a number of physiological and biochemical parameters. A valid and useful toxicokinetic model must have reliable physiological and enzymological data for BD biotransformation before it can be credibly used for human risk assessment.

A physiological model for ligand-induced accumulation of alpha 2u globulin in male rat kidney: roles of protein synthesis and lysosomal degradation in the renal dosimetry of 2,4,4-trimethyl-2-pentanol.

A physiologically based pharmacokinetic (PBPK) model was constructed for the disposition of 2,4,4-trimethyl-2-pentanol (TMP-2-OH) in male rats and its induction of accumulation of renal alpha2u-globulin (alpha2u). The model included diffusion-restricted delivery of TMP-2-OH to compartments representing liver, lung, fat, kidney, GI tract, aggregated rapidly perfused tissues, and aggregated slowly perfused tissues. Metabolism by oxidation and glucuronidation was included for liver and kidneys. Rates of hepatic alpha2u production and resorption by renal proximal tubules were taken from the literature. Degradation of liganded alpha2u by renal lysosomal cathepsins was modeled with a Km value corresponding to the measured 30% reduction in proteolytic efficiency and with free and bound forms of alpha2u competing for access to the enzymes. Increased pinocytotic uptake of alpha2u into the kidney induces cathepsin activity. A model that ascribed renal alpha2u accumulation solely to reduced lysosomal proteolysis failed to reproduce the observed accumulation. The model could reproduce experimental observations if a transient increase in hepatic synthesis of alpha2u, stimulated by the presence of liganded alpha2u in the blood, and accelerated secretion of the protein from the liver were assumed. This model reproduces time course data of blood and kidney TMP-2-OH and renal alpha2u concentrations, suggesting that renal accumulation of alpha2u is not simply a consequence of reduced proteolytic degradation but may also involve a transient increase in hepatic alpha2u production. The model predicts increased delivery of TMP-2-OH to the kidney and consequent increased renal production of potentially toxic TMP-2-OH metabolites than would be the case if no alpha2u were present. Induced lysosomal activity and increased production of toxic metabolites may both contribute to the nephrotoxicity observed in male rats exposed to an alpha2u ligand or its precursor.

Physiological modeling of a proposed mechanism of enzyme induction by TCDD.

A physiological model was previously constructed to facilitate extrapolation of surrogates for the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver to doses comparable to human environmental exposures. The model included induction of P450 isozymes and suggested the presence of multiple binding sites with different affinities for the TCDD-liganded Ah receptor at CYP1A1 dioxin responsive elements. The model also indicated that protein synthesis on the mRNA template exhibited saturation kinetics with respect to message levels. In the present work the earlier model was revised to include the increased proteolysis of the Ah receptor on binding TCDD, more realistic representations of gene transcription and mRNA translation, and different stability for each mRNA. The revised model includes multiple TCDD-liganded Ah receptor binding sites for CYP1A1 and CYP1B1 genes, a lag of 0.2 day for production of mRNA and induced proteins, and stabilization of mRNA by a poly(A) tail. The model reproduced the transient depletion of the Ah receptor subsequent to binding ligand and the dose-response of the receptor in rats treated with biweekly oral doses of TCDD in corn oil. The model reproduced tissue TCDD concentrations observed for several dosing scenarios. Such robustness indicates the utility of the model in estimating internal dose. The model also reproduced the observed dose-response patterns for mRNA and protein for CYP1A1, CYP1A2, and CYP1B1 after repeated dosing. Neither of the two dissociation constants for the Ah receptor bound to the CYP1B1 gene is negligible, supporting the assumption of multiple response elements for this gene. The poorer induction of CYP1B1 was predicted to be due to lower affinity of the dioxin responsive elements for binding the liganded Ah receptor, suggesting the involvement of other regulatory factors, and a shorter poly(A) tail on CYP1B1 mRNA, leading to a shorter lifetime. Saturation in the kinetics of protein synthesis was linked to the limited number of ribosomes that could bind to each message molecule, resulting in fewer ribosomes bound per message at higher doses. Predicted induction at low doses was found to vary widely with the assumptions used in the construction of a model. More detailed descriptions of biological processes might provide more reliable predictions of enzyme induction.

Physiological modeling of butadiene disposition in mice and rats.

The earliest physiological models of 1,3-butadiene disposition reproduced uptake of the gas from closed chambers but over-predicted steady-state circulating concentrations of the mutagenic intermediates 1,2-epoxybut-3-ene and 1,2:3,4-diepoxybutane. A preliminary model based on the observation of a transient complex between cytochrome P450 and microsomal epoxide hydrolase on the endoplasmic reticulum membrane reproduced the blood epoxide concentrations as well as the chamber uptake data. This model was enhanced by the addition of equations for the production and detoxication of 3,4-epoxybutane-1,2-diol in the liver, lungs, and kidneys. The model includes flow-restricted delivery of butadiene and its metabolites to compartments for lungs, liver, fat, kidneys, gastrointestinal tract, other rapidly perfused tissues, and other slowly perfused tissues. Blood was distributed among compartments for arterial, venous, and tissue capillary spaces. Channeling of the three bound epoxides to epoxide hydrolase and their release from the endoplasmic reticulum are competing processes in this model. Parameters were estimated to fit data for chamber uptake of butadiene and epoxybutene, steady-state blood concentrations of epoxybutene and diepoxybutane, and the fractions of the inhaled dose of butadiene that appears as various excreted metabolites. The optimal values of the apparent K(m)s of membrane-bound epoxides for epoxide hydrolase were only 5% of the values for the cytosolic substrate, consistent with the observation of a transient complex between epoxide hydrolase and the cytochrome P450 that produces the epoxide. This proximity effect corresponds to the notion that epoxides produced in situ have privileged access to epoxide hydrolase. The model also predicts considerable accumulation of epoxybutanediol, in agreement with the observation that most of the DNA adducts in animals exposed to butadiene arise from this metabolite.

Achieving credibility in risk assessment models.

Validation of a mathematical model requires demonstrating that a model is free of mathematical errors (internal consistency), is sensitive to large but not small errors or uncertainties in parameter values (verifiability and robustness), reproduces experimental observations on the system being modeled (external consistency), and leads to testable predictions of the system's biological properties. To be heuristically valid, a model also must be a realistic representation of the actual biological system. Only then would the model's predictions be credible to the wider community of biological scientists who would use the model for risk assessment and dose or species extrapolation. Owing to incomplete data, most current dosimetric models are insufficiently realistic to pass this test of credibility. Enhancements to such models that would help achieve credibility are presented, and suggestions are offered for institutionalizing realistic modeling practices in risk assessment.

Propagation of information in MetaNet graph models.

Information flow in metabolic networks has been studied with a graph model which represents the biochemical transformations occurring in the system under investigation. The "signal strength", an algebraic expression which estimates the probability that an intermediate metabolite is bound to a given enzyme, has been used to derive the "signal transmittance", the fraction of the informational signal at one intermediate that reaches another intermediate. The transmittance has been used to derive the "response ratio", the sensitivity of the rate of change of information at one metabolite consequent to a perturbation at another metabolite. Because the graphical representation corresponds to the biochemical events presumed to occur in the network, these quantities can be used to design experiments to confirm or falsify the hypotheses underlying the model and aid in understanding the regulatory properties of the system. The technique is illustrated by an example model, and its predictions are shown to be sensitive to modest structural changes in the network.

Computer simulation of metabolism in palmitate-perfused rat heart. III. Sensitivity analysis.

The behavior of a computer model of metabolism in glucose- and palmitate-perfused rat hearts was interpreted by sensitivity analysis to explain why the heart preferentially utilizes fatty acids as fuel even in the presence of substantial exogenous glucose. The sensitivity functions identified those metabolites and enzymes which were most important in regulating the metabolic rate and determined which enzymes set the levels of the critical metabolites. Control of the mitochondrial redox potential and the distribution of coenzyme A thioesters regulated the rate of fatty acid utilization while strong inhibition of citrate synthetase resulted in accumulation of acetyl CoA and suppression of pyruvate oxidation. Glycolysis was limited by the cytosolic ATP/ADP ratio set largely by the creatine shuttle. Metabolic control appears to be widely distributed rather than localized at "key" enzymes. Metabolite levels are usually set by enzymes controlled by modifiers whereas metabolic flux is regulated by the enzymes that produce ligands for the modifier-controlled enzymes.

Effects of TCDD on thyroid hormone homeostasis in the rat.

A physiological dosimetric model was constructed to describe the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on circulating thyroid hormones in the rat and to test the hypothesis that these hormonal changes cause chronically elevated serum thyrotropin (thyroid stimulating hormone, TSH), which mediates growth promotion and may lead to thyroid tumors in TCDD-treated rats. The model included diffusion restricted distribution of TCDD among compartments for liver, kidney, white fat, slowly and rapidly perfused tissues, and the thyroxine-sensitive tissues brown fat, pituitary, and thyroid. Blood was distributed among major vessels and the capillary beds of the tissues. Metabolism of TCDD was limited to the liver. Secretion of 3,5,3'-triiodothyronine (T3) and thyroxine (3,5,3',5'-tetraiodothyronine, T4) from the thyroid was modeled as stimulated by circulating TSH, whose release from the pituitary was regulated by the hypothalamic peptides thyrotropin releasing hormone (activating) and somatostatin (inhibiting). Release of these peptides was represented as inhibited and activated, respectively, by circulating T4. Binding proteins for T3 and T4 and metabolism of the hormones by deiodination were included in thyroxine-sensitive tissues. Induction of hepatic UDP-glucuronosyltransferase-1*6 (UGT), the enzyme which glucuronidates T4, was modeled as induced by the complex formed between TCDD and the aryl hydrocarbon receptor. The computed extent of deiodination, primacy of the thyroid in generating T3 from T4, dependence of liver and kidney on locally produced T3, and export of T3 formed in the pituitary agreed with experimental observations. The model reproduced the observed decrease in circulating T4 and elevated serum TSH following chronic administration of TCDD. The altered levels were attributed to the increased clearance of T4 by the induced UGT and the consequent modification of feedback control of hormone releases. These results are consistent with the hypothesis of growth stimulation by elevated TSH, but measured values of this hormone in blood of rats vary over a large range, and the change induced by TCDD is often small. Measured UGT levels are less variable and the increase in this protein is much greater, suggesting that this response may be a more reliable biomarker for effects of TCDD on the thyroid.

The importance of anatomical realism for validation of physiological models of disposition of inhaled toxicants.

The goodness of fit of three PBPK models to data for inhalation uptake of 1,3-butadiene by mice from closed chambers were compared. These models included a classical flow-limited model with blood consolidated into arterial and venous compartments, a flow-limited model with implicit blood and alveolar compartments, and a model with an actual alveolar compartment and blood distributed among compartments for arterial, venous, and capillary spaces. Using physiological and biochemical parameters from the literature, all three models reproduced observed steady-state blood butadiene concentrations. However, the first two models predicted more rapid uptake of butadiene than was observed. Assumptions such as ignoring extrahepatic metabolism or reducing the ventilation rate by 75% were required to enable these models to fit the butadiene uptake data. The behavior of the third model resembled that of the other two models when the single-pass extraction ratio of butadiene for all tissues was close to 1, but the model did reproduce observed butadiene uptake when an extraction ratio of 0.5 was used. The difference in predictions of the three models was traced to smaller computed blood:tissue gradients and tissue butadiene concentrations, hence reduced rates of metabolic clearance, when the blood is distributed. These results suggest that the common assumption of flow limitation in the disposition of an inhaled gas may not always be appropriate. Because structurally different models can reproduce the same uptake data and all these models cannot be correct, the assumptions on which these models were based must be investigated experimentally to ensure that they are physiologically realistic.

Species differences in the production and clearance of 1,3-butadiene metabolites: a mechanistic model indicates predominantly physiological, not biochemical, control.

Inhaled 1,3-butadiene, a monomer used in the production of synthetic rubber and other resins, is metabolized to mutagenic and carcinogenic epoxide intermediates. A physiologically based pharmacokinetic model of the uptake, tissue distribution, and metabolism of butadiene was constructed to determine if the biochemical kinetic constants obtained from in vitro studies are consistent with the observed in vivo uptake and metabolism. The model includes compartments for lung, blood, fat, liver, other rapidly perfused tissues ('viscera') and slowly perfused tissues. Metabolism of butadiene was assumed to occur in viscera in addition to lung and liver. Enzymatic reaction rate equations for the formation of 1,2-epoxy-3-butene, for hydrolysis of this epoxide, and for its conjugation with glutathione were also included. Physiological and biochemical parameters for the mouse, rat and human were obtained from the literature; they were not adjusted to produce a fit to experimental data. The model was used to test the hypothesis that differences in uptake and clearance of butadiene by the three species are due to differences in the activities of the metabolizing enzymes. The model reproduces whole-body observations for the mouse and rat. It predicts that inhalation uptake of butadiene and formation and retention of epoxybutene are controlled to a much greater extent by physiological parameters than by biochemical parameters and that storage in the fat represents a significant fraction of the retained butadiene. Accumulation of epoxybutene in the blood is predicted to be higher in mice than in rats or humans, but accumulation of the epoxide intermediate in the liver is predicted to be highest in humans. The epoxide tissue concentrations predicted by the model do not, by themselves, correlate with tumor incidence in mice and rats, indicating that other factors are crucial for carcinogenesis induced by butadiene.