Ranges of phenotypic flexibility in healthy subjects.

BACKGROUND: A key feature of metabolic health is the ability to adapt upon dietary perturbations. A systemic review defined an optimal nutritional challenge test, the "PhenFlex test" (PFT). Recently, it has been shown that the PFT enables the quantification of all relevant metabolic processes involved in maintaining or regaining homeostasis of metabolic health. Furthermore, it was demonstrated that quantification of PFT response was more sensitive as compared to fasting markers in demonstrating reduced phenotypic flexibility in metabolically impaired type 2 diabetes subjects. METHODS: This study aims to demonstrate that quantification of PFT response can discriminate between different states of health within the healthy range of the population. Therefore, 100 healthy subjects were enrolled (50 males, 50 females) ranging in age (young, middle, old) and body fat percentage (low, medium, high), assuming variation in phenotypic flexibility. Biomarkers were selected to quantify main processes which characterize phenotypic flexibility in response to PFT: flexibility in glucose, lipid, amino acid and vitamin metabolism, and metabolic stress. Individual phenotypic flexibility was visualized using the "health space" by representing the four processes on the health space axes. By quantifying and presenting the study subjects in this space, individual phenotypic flexibility was visualized. RESULTS: Using the "health space" visualization, differences between groups as well as within groups from the healthy range of the population can be easily and intuitively assessed. The health space showed a different adaptation to the metabolic PhenFlex test in the extremes of the recruited population; persons of young age with low to normal fat percentage had a markedly different position in the health space as compared to persons from old age with normal to high fat percentage. CONCLUSION: The results of the metabolic PhenFlex test in conjunction with the health space reliably assessed health on an individual basis. This quantification can be used in the future for personalized health quantification and advice.

Hepatic insulin resistance both in prediabetic and diabetic patients determines postprandial lipoprotein metabolism: from the CORDIOPREV study.

BACKGROUND/AIMS: Previous evidences have shown the presence of a prolonged and exaggerated postprandial response in type 2 diabetes mellitus (T2DM) and its relation with an increase of cardiovascular risk. However, the response in prediabetes population has not been established. The objective was to analyze the degree of postprandial lipemia response in the CORDIOPREV clinical trial (NCT00924937) according to the diabetic status. METHODS: 1002 patients were submitted to an oral fat load test meal (OFTT) with 0.7 g fat/kg body weight [12 % saturated fatty acids (SFA), 10 % polyunsaturated fatty acids (PUFA), 43 % monounsaturated fatty acids (MUFA), 10 % protein and 25 % carbohydrates]. Serial blood test analyzing lipid fractions were drawn at 0, 1, 2, 3 and 4 h during postprandial state. Postprandial triglycerides (TG) concentration at any point >2.5 mmol/L (220 mg/dL) has been established as undesirable response. We explored the dynamic response in 57 non-diabetic, 364 prediabetic and 581 type 2 diabetic patients. Additionally, the postprandial response was evaluated according to basal insulin resistance subgroups in patients non-diabetic and diabetic without pharmacological treatment (N = 642). RESULTS: Prevalence of undesirable postprandial TG was 35 % in non-diabetic, 48 % in prediabetic and 59 % in diabetic subgroup, respectively (p < 0.001). Interestingly, prediabetic patients displayed higher plasma TG and large triacylglycerol-rich lipoproteins (TRLs-TG) postprandial response compared with those non-diabetic patients (p < 0.001 and p = 0.003 respectively). Moreover, the area under the curve (AUC) of TG and AUC of TRLs-TG was greater in the prediabetic group compared with non-diabetic patients (p < 0.001 and p < 0.005 respectively). Patients with liver insulin resistance (liver-IR) showed higher postprandial response of TG compared with those patients with muscle-IR or without any insulin-resistance respectively (p < 0.001). CONCLUSIONS: Our findings demonstrate that prediabetic patients show a lower phenotypic flexibility after external aggression, such as OFTT compared with nondiabetic patients. The postprandial response increases progressively according to non-diabetic, prediabetic and type 2 diabetic state and it is higher in patients with liver insulin-resistance. To identify this subgroup of patients is important to treat more intensively in order to avoid future cardiometabolic complications.

EURRECA: development of tools to improve the alignment of micronutrient recommendations.

Approaches through which reference values for micronutrients are derived, as well as the reference values themselves, vary considerably across countries. Harmonisation is needed to improve nutrition policy and public health strategies. The EURRECA (EURopean micronutrient RECommendations Aligned, http://www.eurreca.org) Network of Excellence is developing generic tools for systematically establishing and updating micronutrient reference values or recommendations. Different types of instruments (including best practice guidelines, interlinked web pages, online databases and decision trees) have been identified. The first set of instruments is for training purposes and includes mainly interactive digital learning materials. The second set of instruments comprises collection and interlinkage of diverse information sources that have widely varying contents and purposes. In general, these sources are collections of existing information. The purpose of the majority of these information sources is to provide guidance on best practice for use in a wider scientific community or for users and stakeholders of reference values. The third set of instruments includes decision trees and frameworks. The purpose of these tools is to guide non-scientists in decision making based on scientific evidence. This platform of instruments will, in particular in Central and Eastern European countries, contribute to future capacity-building development in nutrition. The use of these tools by the scientific community, the European Food Safety Authority, bodies responsible for setting national nutrient requirements and others should ultimately help to align nutrient-based recommendations across Europe. Therefore, EURRECA can contribute towards nutrition policy development and public health strategies.

Protein adduct formation by glucuronide metabolites of permethrin.

Biomonitoring of exposure to the insecticide permethrin is usually performed by analysis of its urinary metabolites 3-phenoxybenzoic acid (3-PBA) or cis/ trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Cl 2 CA). We are engaged in the development of a methodology to assess the cumulative internal dose of exposure to permethrin, which is based on the assumption that (reactive) glucuronide conjugates of the major permethrin metabolites 3-PBA and Cl 2 CA will form persistent (weeks to months) adducts to proteins, in analogy with the glucuronide conjugates of structurally related drugs. The 3-PBA and Cl 2 CA beta-glucuronide metabolites of permethrin have been successfully chemically and enzymatically synthesized. Their identities have been assessed by means of (1)H NMR spectroscopy and liquid chromatography-tandem mass spectrometry. The reactivity of these metabolites with various amino acids, peptides, and albumin in human plasma has been studied. Several distinct adducts could be identified by liquid chromatography-tandem mass spectrometry. After pronase digestion of albumin isolated from exposed human plasma, various lysine derivatives resulted with favorable mass spectrometric and chromatographic properties. Covalent binding was quantified by using [(14)C]-3-PBA glucuronide; >1.5% of total radioactivity was bound to proteins. It is envisaged that the obtained results can form a firm basis for the development of a protein adduct-based methodology for biomonitoring exposure to permethrin. In view of the widespread use of permethrin, the toxicological relevance of protein binding by its metabolites will be addressed in more detail in future work.

Expression profiling of colon cancer cell lines and colon biopsies: towards a screening system for potential cancer-preventive compounds.

Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived from colonic tissue were measured using cDNA microarrays with 4000 genes and compared with expression profiles in biopsies of human colon tumours and normal tissue. Differences and similarities in the gene expression profiles of the cell lines were analysed by clustering and principal component analysis (PCA). Cytoskeleton genes and immune response genes are two functional classes of genes that contributed to the differences between the cell lines. A subset of 72 colon cancer-specific genes was identified by comparing expression profiles in human colon biopsies of tumour tissue and normal tissue. A separation of the cell lines based on the tumour stage of the original adenocarcinoma was observed after PCA of expression data of the subset of colon cancer-specific genes in the cell lines. The results of this study may be useful in the ongoing research into mechanisms of cancer prevention by dietary components.

Disposition of 14C-alpha-cyclodextrin in germ-free and conventional rats.

The absorption, disposition, metabolism, and excretion of uniformly (14)C-labeled alpha-cyclodextrin ((14)C-alpha-CD) was examined in four separate experiments with Wistar rats. In Experiment 1, (14)C-alpha-CD (25 microCi, 50 mg/kg bw) was administered intravenously to four male and four female conventional rats. In Experiment 2, (14)C-alpha-CD (25 microCi, 200 mg/kg bw) was given by gavage to four male and four female germ-free rats. In Experiments 3 and 4, (14)C-alpha-CD was given to groups of four male and four female conventional rats by gavage at different dose levels (100 microCi, 200 mg/kg bw; 25 microCi, 200 and 100 mg/kg bw). In all experiments, (14)C was measured in respiratory CO(2), urine, and feces over periods of 24-48 h, and in the contents of the gastrointestinal tract, blood, main organs, and residual carcass at termination of the experiments. The chemical identity of the (14)C-labeled compounds was examined by HPLC in blood (Experiment 1), urine (Experiments 1-4), feces (Experiments 2-4), and samples of intestinal contents (Experiments 2 and 4). Recovered (14)C was expressed as percentage of the administered dose. Experiment 1 showed that intravenously administered alpha-CD is excreted rapidly with urine. During the first 2h after dosing, plasma (14)C levels decreased rapidly (t(1/2), 26 and 21 min in male and female rats, respectively). About 13% of the administered (14)C dose (range 4.6-30.6) was detected in the feces, respiratory CO(2), organs, and carcass at the end of the experiment, i.e., 24 h after dosing. The presence of about 1.9% in the intestinal contents and feces suggests that a certain fraction of systemic alpha-CD is eliminated with the bile or saliva. Conclusive evidence, either positive or negative, for a hydrolysis and further metabolism of a small fraction of the administered alpha-CD by the enzymes of the mammalian body could not be gained from this experiment. Upon oral administration of (14)C-alpha-CD to germ-free rats (Experiment 2), about 1.3% of the label expired as CO(2) within 24 h. In the urine collected from 0 to 8 h after dosing, (14)C-alpha-CD was the only radiolabeled compound detected. The amounts of alpha-CD detected in the urine suggest that on average about 1% of an oral dose is absorbed in rats during small-intestinal passage. In conventional rats (Experiments 3 and 4), a delayed appearance of respiratory (14)CO(2) was observed which is attributed to the non-digestibility of alpha-CD and its subsequent microbial fermentation in the cecum and colon. In the urine collected at 4 h after dosing, a small amount of unchanged (14)C-alpha-CD was detected which confirms that about 1% of the ingested alpha-CD is absorbed intact and is excreted via the kidneys. No (14)C-alpha-CD was found in the feces. It is concluded from the data that ingested (14)C-alpha-CD is not digested in the small intestine of rats but is fermented completely by the intestinal microbiota to absorbable short-chain fatty acids. Therefore, the metabolism of alpha-CD resembles closely that of resistant starch or other fermentable dietary fibers.

Antimutagenic activity of green tea and black tea extracts studied in a dynamic in vitro gastrointestinal model.

An in vitro gastrointestinal model, which simulates the conditions in the human digestive tract, was used to determine potential antimutagenic activity of extracts of black tea and green tea. In this paper, results are presented on the availability for absorption of potential antimutagenic compounds present in tea and on the influence of the food matrix on this activity. Between 60 and 180min after the tea was introduced into the model, antimutagenic activity was recovered from the jejunal compartment by means of dialysis: the dialysate appeared to inhibit the mutagenicity of the food mutagen MeIQx in the direct plate assay with Salmonella typhimurium (Ames test). The maximum inhibition was measured at 2h after the start of the experiment and was comparable for black tea and green tea extract. To determine the influence of food matrices on the antimutagenic activity of tea, the model was loaded with black tea together with milk or a homogenized standard breakfast. The maximum inhibition observed with black tea was reduced by 22, 42 and 78% in the presence of whole milk, semi-skimmed milk, and skimmed milk, respectively. Whole milk and skimmed milk abolished the antimutagenic activity of green tea by more than 90%; for semi-skimmed milk the inhibition was more than 60%. When a homogenized breakfast was added into the model together with the black tea extract, the antimutagenic activity was completely eliminated. When tea and MeIQx were added together into the digestion model, MeIQx mutagenicity was efficiently inhibited, with green tea showing a slightly stronger antimutagenic activity than black tea. In this case, the addition of milk had only a small inhibiting effect on the antimutagenicity. Antioxidant capacity and the concentration of catechins were also measured in the jejunal dialysates. The reduction in antimutagenic activity corresponded with reduction in antioxidant capacity and with a decrease of concentration of three catechins, viz. catechin, epigallocatechin gallate and epigallocatechin. The in vitro gastrointestinal model appears to be a useful tool to study the antimutagenicity of food components.

Functional food ingredients against colorectal cancer. An example project integrating functional genomics, nutrition and health.

Functional Food Ingredients Against Colorectal Cancer is one of the first European Union funded Research Projects at the cross-road of functional genomics [comprising transcriptomics, the measurement of the expression of all messengers RNA (mRNAs) and proteomics, the measurement of expression/state of all proteins], nutrition and human health. The goal of Functional Food Ingredients Against Colorectal Cancer is to develop a colon epithelial cell line-based screening assay for nutrients with presumed anti-colorectal carcinogenic properties. Genes involved in colon carcinogenesis are identified at the RNA and protein level, using a variety of methods (subtractive hybridisation, DNA microarray, proteomics) in combination with models for colorectal cancer development (human biopsies, rat model for colorectal carcinogenesis, colorectal cancer epithelial cell lines). Secondly, colorectal cancer epithelial cell lines are selected, in terms of their capacity to undergo gene/protein expression changes representing different phases in the colorectal carcinogenesis. Thirdly, these cell lines are used to determine the effects of nutrients with presumed anti-carcinogenic properties (e.g. resveratrol, flavonoids) on functional genomics-derived endpoints. Once validated against the effects of these nutrients in in vivo animal models and classical biomarkers for colorectal carcinogenesis, these cell line models combined with functional genomics represent useful tools to study colorectal carcinogenesis and screen for nutrients with anti-carcinogenic properties.

Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats.

The absorption, disposition, metabolism, and excretion of 14C-labeled gamma-cyclodextrin ([14C]gamma-CD) was examined in four separate experiments with Wistar rats. In experiment 1, [14C]gamma-CD (25 microCi, 600 mg/kg body wt) was administered intravenously to four male and four female conventional rats. In experiment 2, [14C]gamma-CD (25 microCi, 1000 mg/kg body wt) was given by gavage to four male and four female germ-free rats. In experiments 3 and 4, [14C]gamma-CD (25 and 100 microCi, respectively, 1000 mg/kg body wt) was given to four male and four female conventional rats by gavage. In all experiments, 14C was measured in respiratory CO2, urine, feces, contents of the gastrointestinal tract, blood, main organs, and residual carcass. The chemical identity of the 14C-labeled compounds was examined by HPLC in urine (experiments 1-4), blood (experiments 1 and 4), and samples of intestinal contents (experiments 3 and 4). Recovered 14C was expressed as a percentage of the administered dose. Total recovery of 14C varied between about 90% (experiments 3 and 4) and 100% (experiments 1 and 2). Experiment 1 showed that about 90% of intravenously administered gamma-CD is excreted in urine within 24 h. During the first 2 h after dosing, plasma 14C levels decreased rapidly (t1/2, 15-20 min). The remaining 10% of the dose was probably excreted into the gastrointestinal tract with bile and saliva. In addition, some [14C]gamma-CD may have been degraded by plasma and tissue amylases. Since glucose is the common product of systemic hydrolysis and intestinal digestion, it is not possible to quantitate the relative flux of [14C]gamma-CD through these two pathways. Upon oral administration of [14C]gamma-CD to germ-free rats (experiment 2), about 66% of the label was expired as CO2 within 24 h. The rate of 14C exhalation reached a maximum at 90 min after dosing and then declined steadily. In the urine, and in the contents of the cecum and colon collected at 24 h, [14C]gamma-CD was not found (except for a trace in the cecum of females). In conventional rats (experiments 3 and 4), a similar, fast appearance of respiratory 14CO2 was observed. There was no delayed formation of 14CO2 due to incomplete digestion and subsequent microbial fermentation in the cecum and colon. In pooled urine collected at 4 h after dosing, a small amount of unchanged [14C]gamma-CD was detected (experiment 4). From this result, it was estimated that less than 0.02% of ingested intact gamma-CD was absorbed and excreted with the urine. It is concluded from the data that ingested [14C]gamma-CD is rapidly and essentially completely digested in the small intestine to absorbable [14C]glucose. The absorption of intact [14C]gamma-CD by passive diffusion is very low (<0.02%). Therefore, the metabolism of gamma-CD resembles closely that of starch or linear dextrins.

A physiologically based pharmacokinetic (PB-PK) model for 1,2-dichlorobenzene linked to two possible parameters of toxicity.

A physiologically based pharmacokinetic (PB-PK) model was developed for 1,2-dichlorobenzene (1,2-DCB) for the rat. This model was adjusted for the human situation, using human in vitro parameters, including a Vmax and Km determined with human microsomes. For comparison, the Vmax and Km values from the rat were scaled allometrically to the human case. The model was used in two ways: (1) Acute hepatotoxicity was related to the amount of reactive metabolites (epoxides) formed in vitro. For rats, the hepatic concentration of epoxide metabolites in vivo after exposure to a toxic dose level (250 mg/kg bw) was predicted using in vitro parameters. For man, the dose level needed to obtain the same toxic liver concentration of reactive metabolites as in rat was predicted, assuming a concentration-effect relationship in the liver. It could be concluded that this concentration is not reached, even after induction of the oxidation step, due to saturation of metabolism and a concomitant accumulation of 1,2-DCB in fat. (2) Hepatotoxicity was related to depletion of glutathione (GSH) in the liver. In the model, the consumption of hepatic GSH by metabolism (based on in vivo and in vitro data) and normal turnover was described. In vivo validation was conducted by comparing the predictions of the model with the results of a GSH depletion study performed at two dose levels (50 and 250 mg/kg bw). Subsequently, the GSH consumption by 1,2-DCB metabolites was estimated for man using human in vitro metabolic data. GSH turnover in human liver was assumed to be the same as that in rat. It appeared that at a dose level of 250 mg/kg, hepatic GSH was completely depleted after 10 hr for man, whereas for the rat a maximum depletion of 75% was predicted, after 15 hr. The presented model provides a quantitative tool for evaluating human risk for two different toxicity scenarios, namely covalent binding of reactive metabolites and depletion of GSH.

Species and strain differences in the hepatic cytochrome P450-mediated biotransformation of 1,4-dichlorobenzene.

Our goal was to characterize possible species and strain differences in the hepatic microsomal biotransformation of 1,4-dichlorobenzene (1,4-DCB). Experiments compared extent of labeled 1,4-DCB conversion to oxidized metabolites, glutathione conjugates, and covalently bound metabolites by hepatic microsomes from humans, from male B6C3F1 mice, and from males of three rat strains (Fischer 344, Sprague-Dawley (SD), and Wistar). These rodents were selected for comparison because of their dissimilar responses to 1,4-DCB, notably, hepatocarcinogenicity in the B6C3F1 mouse but not the Wistar or Fischer rat, and nephrotoxicity and carcinogenicity in the Fischer rat. The species rank order for total in vitro conversion of 1,4-DCB was mouse > rat >> human. Conversion by microsomes from Fischer and Wistar rats was similar, whereas SD rats showed less biotransformation than the other two strains. Microsomes from the mouse produced most of the reactive metabolites as indicated by covalent binding to macromolecules (>20% of total metabolites formed). This covalent binding by mouse microsomes was extensively inhibited by ascorbic acid (AA), with a concomitant increase in hydroquinone formation, indicating an important role for benzoquinones as reactive metabolites. Phenobarbital pretreatment of rats enhanced the in vitro conversion of 1,4-DCB and the amount of covalent binding. Covalent binding for all rat microsomes was partly (33-79%) inhibited by AA. Addition of glutathione (GSH) plus AA further diminished the covalent binding with concomitant increased formation of the GSH-conjugated epoxide. Human microsomes produced the least reactive metabolites, with the majority (>70%) of this covalent binding prevented by GSH addition. The observed species differences, notably the more pronounced biotransformation of 1,4-DCB to reactive species including benzoquinones, could be factors in this compound's liver carcinogenicity in B6C3F1 mice but not other rodent species.

Kinetics and metabolism of 1,4-dichlorobenzene in male Wistar rats: no evidence for quinone metabolites.

The biotransformation and kinetics of 1,4-dichlorobenzene (1,4-DCB) were studied in male Wistar rats at three oral dose levels (10, 50 and 250 mg/kg). The effect of induction of CYP2E1 by isoniazid on the kinetics and biotransformation was determined. Excretion was predominantly via the urine (78-85%) and to a small extent via the faeces (2-5%). The relative contributions of these routes were not dose dependent. Excretion via the bile ranged from less than 5% at the low dose level to 30% at the high dose level. The major biliary metabolite was the glucuronide of 2,5-dichlorophenol (2,5-DCP). The time point at which the plasma concentrations of the parent compound and the metabolites were maximal (TCmax) as well as the maximum concentrations (Cmax) increased with higher dose level. Induction by isoniazid resulted in a faster urinary elimination, whereas TCmax and Cmax were lower for induced rats. In addition, the area under the blood curve (AUC) was smaller and total clearance was higher for induced rats. 1,4-DCB was mainly metabolized to 2,5-DCP (ca. 90%), which was detected in the urine as its sulfate (50-60%), glucuronide (20-30%) and the free form (5-10%). Minor metabolites were the N-acetyl-cysteine-S-dihydro-hydroxy-1,4-dichlorobenzene and the corresponding dehydrated N-acetyl-cysteine-S-1,4-dichlorobenzene, which comprised ca. 10% of total metabolites. No hydroquinones were observed for the male Wistar rat, not even under conditions of induced oxidative metabolism.

Differences in cytochrome P450-mediated biotransformation of 1,2-dichlorobenzene by rat and man: implications for human risk assessment.

The oxidative biotransformation of 1,2-dichlorobenzene (1,2-DCB) was investigated using hepatic microsomes from male Wistar, Fischer-344 and Sprague-Dawley (SD) rats, phenobarbital (PB)- and isoniazid (ISO) pretreated male Wistar rats and from man. In addition, microsomes from cell lines selectively expressing one cytochrome P450 (P4502E1, 1A1, 1A2, 2B6, 2C9, 2D6, /A6 and 3A4) were used. The rate of conversion was 0.09 nmol/min/mg. protein for both Wistar and Fischer-344 rat microsomes, 0.04 for SD-microsomes and 0.14 for human microsomes. Induction of Wistar rats with isoniazid (ISO, a P4502E1 inducer) or phenobarbital (PB, a P4502B1/2 inducer) resulted in an increased conversion rate of 0.20 and 0.42 nmol/min/mg. protein, respectively. Covalent binding of radioactivity to microsomal protein was similar for Wistar, Fischer and ISO-pretreated rats (16-17% of total metabolites), whereas induction with PB resulted in an increased covalent binding of 23% of total metabolites. Covalent binding was 31% for SD-microsomes and only 4.6% for human microsomes. Ascorbic acid notably reduced the amount of covalently bound metabolites for the SD-microsomes only, indicating that for these microsomes quinones were likely to be involved in this part of the covalent binding. Conjugation of epoxides with glutathione (GSH) inhibited most of the covalent binding for all microsomes. In the absence of GSH, the epoxides were hydrolyzed by epoxide hydrolase, resulting in the formation of dihydrodiols. Inhibition of epoxide hydrolase resulted in a decreased conversion and an increased covalent binding for all microsomes tested, indicating a role of epoxides in the covalent binding. Fischer-344 rat liver microsomes showed a lower epoxide hydrolase activity than microsomes from Wistar and Sprague-Dawley rats, which may explain the higher sensitivity to 1,2-DCB induced hepatotoxicity of Fischer rats in vivo. Conjugation of the epoxides with GSH was predominantly non-enzymatic for the rat, whereas for man, conjugation was nearly exclusively catalyzed by glutathione-S-transferases. This difference may be explained by the formation of a 'non-reactive' 3,4-epoxide by P4502E1 in human microsomes: incubations with microsomes selectively expressing human P4502E1 as well as human liver microsomes, resulted in the formation of similar amounts of 2,3- and 3,4-dichlorophenol (DCP), as well as two GSH-epoxide conjugates in equal amounts. For rat microsomes, one major GSH-epoxide conjugate was found, and a much higher covalent binding, particularly for the PB-microsomes. Therefore, we postulate that rat P4502B1/2 preferentially oxidizes the 4,5-site of 1,2-DCB, resulting in a reactive epoxide. Postulating these epoxides to be involved in the mechanism(s) of toxicity, human risk after exposure to 1,2-DCB will be overestimated when risk assessment is solely based on toxicity studies conducted in rat.

Disposition of 14C-erythritol in germfree and conventional rats.

The metabolism and disposition of U-14C-erythritol was examined in four groups of three male and three female, nonfasted rats each. The rats of groups A and D were germfree; the rats of groups B and C were kept under conventional conditions. The rats of group B received an erythritol-supplemented diet for 3 weeks prior to the experiment (adapted rats). The rats of groups A, C, and D were kept on an ordinary diet which was sterile for groups A and D (not adapted rats). On the day of the experiment, each rat was dosed with U-14C-erythritol by gavage (5 microCi/kg body wt; sp act 50 microCi/g erythritol). The radiochemical purity of the erythritol was 96.43% for groups A-C. Group D, which was attached to the study after evaluation of the results of groups A-C, received a more purified erythritol with a radiochemical purity of 99.46% because the data of group A pointed to a possible interference by a 14C-labeled impurity in the commercial 14C-erythritol. After dosing, respiratory CO2 and urine were collected from each rat at regular intervals for 24 hr. At termination, feces were also collected. The animals were killed and intestinal contents, organs, tissues, and the remaining carcass processed for determination of 14C-14C was excreted rapidly in the urine of all groups (range of groups A-D: 47.3-60.6% of the administered dose within the first 4 hr). Total 24-hr urinary excretion varied between 67.0% (group B) and 81.4% (group D). HPLC analysis of the urine showed that more than 96% of the eluted radiolabel represented erythritol. Conventional, adapted rats expired more 14CO2 than conventional, unadapted rats [10.9% (B) vs 6.7% (C)]. Germfree rats expired much less 14CO2 [0.8% (A) and 0.3% (D)]. In germfree rats, 14CO2 expiration started shortly after dosing, reaching half of the 24-hr excretion after about 2.5 hr. In conventional rats 14CO2 expiration started with a delay of about 2 hr reaching half the 24-hr excretion after 4-6 hr. The excretion of 14C with feces was similar in all groups (8.3% on average of all rats). Slightly more 14C was retained in the intestinal contents of germfree than conventional rats (1.9 vs 0.5%). The body retention was higher in conventional than in germfree rats (3.4 vs 2.0%). In group D, body retention was lowest (1.6%). The total recovery of 14C was similar in all groups (95.6%, average of all rats). It is concluded that ingested erythritol is efficiently absorbed mainly from the small intestine, is not metabolized to a relevant extent in the body, and is excreted unchanged in the urine. The fraction of erythritol not absorbed is fermented by the gut microflora to intermediate products which are largely absorbed and metabolized. The data support a proposed physiological energy value for erythritol of about 0.5 kcal/g.

Regioselectivity and quantitative structure-activity relationships for the conjugation of a series of fluoronitrobenzenes by purified glutathione S-transferase enzymes from rat and man.

Quantitative structure-activity relationships (QSAR's) are described for the rate of conjugation of a series of fluoronitrobenzenes with cytosolic as well as with two major alpha and mu class enzymes of rat and human liver, viz., glutathione S-transferases (GST) 1-1, 3-3, A1-1, and M1a-1a. For all purified enzymes studied, the natural logarithm of the rate of conversion of the fluoronitrobenzenes correlates with both the calculated reactivity of the fluoronitrobenzenes for an electrophilic attack (i.e., E(LUMO)) and the calculated relative heat of formation for formation of the respective Meisenheimer complex intermediate (delta delta HF). In addition, the regioselectivity of the reaction was determined and compared. The results obtained strongly support the conclusion that chemical reactivity of the fluoronitrobenzenes is the main factor determining the outcomes of their conversion by all glutathione S-transferase enzymes. The regioselectivities vary only a few percent from one enzyme to another, whereas QSAR lines for all purified enzymes are in the same region and run parallel. This indicates that in the overall reaction the nucleophilic attack of the thiolate anion on the fluoronitrobenzenes, leading to formation of the Meisenheimer complex, is the rate-limiting step in the overall catalysis. The fact that chemical reactivity of the fluoronitrobenzenes is the main factor in setting the outcomes of the overall conversion by the different glutathione S-transferase enzymes implies that extrapolation from rat to results of other species including man, and also from one individual to another, must be feasible. That this is actually the case is clearly demonstrated by the results of the present study.

Enzyme kinetics and substrate selectivities of rat glutathione S-transferase isoenzymes towards a series of new 2-substituted 1-chloro-4-nitrobenzenes.

1. Four different rat glutathione S-transferase (GST) isoenzymes, belonging to three different classes, were examined for their GSH conjugating capacity towards 11 2-substituted 1-chloro-4-nitrobenzene derivatives. Significant differences were found in their enzyme kinetic parameters Km, kcat and kcat/Km. 2. Substrates with bulky substituents on the ortho-position appeared to have high affinities (low Km's) for the active site of the GST-isoenzymes, suggesting that there is sufficient space in this area of the active site. A remarkably high Km (low affinity) was found for 2-chloro-5-nitropyridine towards all GST-isoenzymes examined. 3. GST 3-3 catalysed the reaction between GSH and the substrates most efficiently (high kcat) compared with the other GST-isoenzymes. Moreover, GST 3-3 showed clear substrate selectivities towards the substrates with a trifluoromethyl-, chlorine- and bromine-substituent. 1-Chloro-2,4-dinitrobenzene and 2-chloro-5-nitrobenzonitrile were most efficiently conjugated by all four GST-isoenzymes examined. 4. When the rate of the conjugation reactions was followed, a linear increase of formation of GS-conjugate could be seen for 2-chloro-5-nitrobenzonitrile during a much longer period of time than for 1-chloro-2,4-dinitrobenzene with all GST-isoenzymes examined. Therefore, it is suggested that 2-chloro-5-nitrobenzonitrile might be recommended as an alternative model substrate in GST-research.

Cytochrome P450 catalyzed metabolism of 1,2-dibromoethane in liver microsomes of differentially induced rats.

The cytochrome P450 (P450) catalyzed oxidation of 1,2-dibromoethane (1,2-DBE) to 2-bromoacetaldehyde (2-BA) was measured in liver microsomes of both control and differentially induced rats. 2-BA formation was quantified by derivatization of 2-BA with adenosine (ADO), resulting in the formation of the highly fluorescent 1,N6-ethenoadenosine (epsilon-ADO), which was measured by HPLC. After microsomal incubation with 1,2DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65 degrees C appeared necessary to ensure efficient formation of epsilon-ADO. Using this optimized derivatization method to quantitate 2-BA formation, the enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA were measured in liver microsomes prepared from untreated rats and rats pretreated with phenobarbital (PB), beta-naphtoflavone (beta NF) and pyrazole (PYR). P450 isoenzymes in PYR- and beta NF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent Vmax- and Km- values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 microns for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in beta NF-induced microsomes, respectively. Due to the non-linear enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA using control and PB-induced microsomes, no proper Vmax- and Km- values could be calculated. However, from Michaelis-Menten plots it was clear that the affinity of P450 isoenzymes for 1,2-DBE in control and PB-induced microsomes was in the same range when compared to beta NF-induced microsomes and thus much lower than the PYR-induced microsomes.

Dose-dependent kinetics and metabolism of 1,2-dichlorobenzene in rat: effect of pretreatment with phenobarbital.

1. Toxicity of halobenzenes has been ascribed mainly to their epoxides, but recent studies with bromobenzene have shown that secondary quinone metabolites are also involved in the alkylation of hepatic proteins. However, the relative contribution of the quinones and the epoxides to the toxicity of halobenzenes is still unclear. In order to investigate the relation between metabolism and toxicity of 1,2-dichlorobenzene (1,2-DCB), the biotransformation, tissue distribution, blood kinetics, and excretion at three different oral dose levels (5, 50 and 250 mg/kg) of the radiolabelled compound were investigated in the male Wistar rat. A toxic dose level (250 mg/kg, as demonstrated by Allis et al. 1992) was included. 2. The major route of elimination (75-85%) was renal excretion. Excretion via the faeces ranged from 19% for the low dose to 7% for the high-dose level. Excretion was nearly complete within 24 h for the low and mid-dose level, and within 48 h for the high-dose level. Pretreatment with phenobarbital resulted in a more rapid excretion for the high-dose level and an overall higher urinary excretion. Biliary excretion was 50-60%, indicating a considerable enterohepatic circulation. 3. Highest concentrations of radioactivity after a low dose were found in fat, liver and kidney at 6 h after administration, and then declined rapidly. 4. The maximum concentration of radioactivity in blood was reached at 6-8 h for the low and mid-dose level, and at 24 h for the high-dose level. The concentration of parent chemical was essentially constant during 3 and 6 h for the mid- and high-dose level respectively, and then declined. 1,2-DCB could only be detected in blood in the first 2 h after administration of the 5-mg/kg dose. 5. The major route of biotransformation was via the glutathione pathway and 60% of the urinary metabolites were mercapturic acids. In addition, the major metabolites in bile were conjugates of glutathione. Other major metabolites in urine were the sulphate conjugates of 2,3- and 3,4-dichlorophenol (DCP). No significant differences in metabolic profiles were observed between the different doses. Induction with phenobarbital resulted in the increased excretion of sulphate conjugates (30% in the induced rat, 20% in the control rat), mainly the conjugate of 3,4-DCP. 6. The mercapturic acids in urine and glutathione conjugates in bile were epoxide-derived, whereas no quinone- or hydroquinone-derived metabolites were observed. Therefore the hepatotoxicity of 1,2-dichlorobenzene is assumed to be related, at least partly, to the presence of the intermediate arene oxide. A high dose of 1,2-DCB will result in depletion of GSH, followed by oxidative stress and possible binding to macromolecules.