The use of toxicokinetic data in preclinical safety assessment: a toxicologic pathologist perspective.

Collection of toxicokinetic data has become a routine practice during the last 15 years in most general toxicity studies on pharma. It enables the correlation of pathological changes with the plasma concentration of drugs and/or their metabolites. This overview summarizes the use of the toxicokinetic data from the perspective of the toxicologic pathologist.

Sensitivity of (1)H NMR analysis of rat urine in relation to toxicometabonomics. Part I: dose-dependent toxic effects of bromobenzene and paracetamol.

(1)H nuclear magnetic resonance (NMR) spectroscopy of rat urine in combination with pattern recognition analysis was evaluated for early noninvasive detection of toxicity of investigational chemical entities. Bromobenzene (B) and paracetamol (P) were administered at five single oral dosages between 2 and 500 mg/kg and between 6 and 1800 mg/kg, respectively. The sensitivity of the proposed method to detect changes in the NMR spectra 24 and 48 h after single dosing was compared with histopathology and biochemical parameters in plasma and urine.

Uniform procedure of (1)H NMR analysis of rat urine and toxicometabonomics Part II: comparison of NMR profiles for classification of hepatotoxicity.

A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition is proposed for early detection of toxicity of investigational compounds in rats. The method is applied to detect toxicity upon administration of 13 toxic reference compounds and one nontoxic control compound (mianserine) in rats. The toxic compounds are expected to induce necrosis (bromobenzene, paracetamol, carbon tetrachloride, iproniazid, isoniazid, thioacetamide), cholestasis (alpha-naphthylisothiocyanate (ANIT), chlorpromazine, ethinylestradiol, methyltestosterone, ibuprofen), or steatosis (phenobarbital, tetracycline).

The incidence of thymic B lymphoid follicles in healthy beagle dogs.

The incidence of thymic B cell lymphoid follicles was retrospectively studied in 62 male and 58 female healthy control beagle dogs (age 11.3 +/- 4.8, range 6 to 23 months).

Intravenous tolerability of an acid vehicle in Sprague Dawley rats and beagle dogs after daily slow bolus injection and infusion for one hour during 14 days.

To assess the tolerability of an acid vehicle to be used in toxicology studies, a low pH aqueous solution containing 16.4 mg/ml of citric acid, 4.2 mg/ml of disodium phosphate, 25 mg/ml of mannitol, adjusted with phosphoric acid/NaOH 1 M to pH 3 was daily administered intravenously to rats and dogs for 14 consecutive days.

Polar molecular surface as a dominating determinant for oral absorption and brain penetration of drugs.

Purpose: To study oral absorption and brain penetration as a function of polar molecular surface area.

Methods: Measured brain penetration data of 45 drug molecules were investigated. The dynamic polar surface areas were calculated and correlated with the brain penetration data.

Interactions of alpha, beta-unsaturated aldehydes and ketones with human glutathione S-transferase P1-1.

In the present study the irreversible inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by alpha, beta-unsaturated aldehydes and ketones was studied. When GSTP1-1 was incubated with a 50-fold molar excess of the aldehydes acrolein (ACR) and 4-hydroxy-2-nonenal (HNE) and the ketones curcumin (CUR) and ethacrynic acid (EA) at 22 degrees C, all of them inactivated GSTP1-1. The remaining activity after 4 h of incubation in all cases was lower than 10%.

Inhibition of glutathione conjugation by glutathione analogues in the perfused rat liver. Effect of esterification on the potency of gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine.

To assess the role of GST's (glutathione S-transferases) in the (de)toxification of their substrates, an in vivo active inhibitor based on the structure of glutathione (GSH), gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine monoethyl ester (Et-R-Hep), was developed. To increase its effectivity, analogues esterified with alkyl chains of varying lengths and one diesterified derivative (DiEt-R-Hep) were synthesized. The unesterified analogue, R-Hep, was also tested.

Urinary thiodiacetic acid. A selective biomarker for the cytochrome P450-catalyzed oxidation of 1,2-dibromoethane in the rat.

1,2-Dibromoethane (1,2-DBE) is a carcinogenic compound that is metabolized both by cytochrome P450 (P450) and glutathione S-transferase (GST) enzymes, and that has been used by us as a model compound to study interindividual variability in biotransformation reactions. In this study, the excretion of thiodiacetic acid (TDA) and S-(2-hydroxyethyl)-N-acetyl-l-cysteine (2-HEMA) were measured in the urine of rats dosed with 1,2-DBE, and experiments were performed to investigate to what extent P450 and GST enzymes contribute to the formation of TDA. To this end, CYP2E1, the main P450 isoenzyme catalyzing the oxidation of 1,2-DBE, was inhibited using disulfiram and diallylsulfide.

The use of human in vitro metabolic parameters to explore the risk assessment of hazardous compounds: the case of ethylene dibromide.

Ethylene dibromide (1,2-dibromoethane, EDB) is metabolized by two routes: a conjugative route catalyzed by glutathione S-transferases (GST) and an oxidative route catalyzed by cytochrome P450 (P450). The GST route is associated with carcinogenicity. An approach is presented to use human purified GST and P450 enzymes to explore the importance of these metabolic pathways for man in vivo.

Inhibition of glutathione S-transferase activity in human melanoma cells by alpha,beta-unsaturated carbonyl derivatives. Effects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid, and trans-2-hexenal.

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied.

Inter-individual variability in the oxidation of 1,2-dibromoethane: use of heterologously expressed human cytochrome P450 and human liver microsomes.

1,2-Dibromoethane (1,2-DBE) is mainly used as an additive in leaded gasoline and as a soil fumigant and it is a suspected carcinogen in humans. In this study, the oxidative bioactivation of 1,2-DBE to 2-bromoacetaldehyde (2-BA) was studied using heterologously expressed human cytochrome P450 (P450) isoenzymes and human liver microsomes. Out of ten heterologously expressed human P450 isoenzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2C9, CYP2C18, CYP3A4 and CYP3A5), only human CYP2A6, CYP2B6 and CYP2E1 metabolized 1,2-DBE, albeit with strongly differing catalytic efficiencies.

In vitro inhibition of rat and human glutathione S-transferase isoenzymes by disulfiram and diethyldithiocarbamate.

The drug disulfiram (DSF, Antabuse) has been used in the therapy of alcohol abuse. It is a potent inhibitor of aldehyde dehydrogenase. Its reduced form, diethyldithiocarbamate (DDTC), and further metabolites show similar activities.

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.

Inhibition of rat, mouse, and human glutathione S-transferase by eugenol and its oxidation products.

The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme tyrosinase. Tyrosinase was used to oxidize eugenol.

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.

Identification of multiple target sites for a glutathione conjugate on glutathione-S-transferase by matrix-assisted laser desorption/ionization mass spectrometry.

A mass spectrometric method providing qualitative site-specific information regarding covalent modification of proteins is described. The method involves comparison of unmodified and modified proteins by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) peptide mapping in combination with site-specific mutagenesis of possible target amino acids. The approach is demonstrated through the mapping of glutathione-S-transferases (GSH transferases) before and after inhibition with the glutathione conjugate 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GSTCBQ).

Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and in the rat in vivo.

Inhibitors of rat and human Alpha- and Mu-class glutathione S-transferases that effectively inhibit the glutathione (GSH) conjugation of bromosulphophthalein in the rat liver cytosolic fraction, isolated rat hepatocytes and in the rat liver in vivo have been developed. The GSH analogue (R)-5-carboxy-2-gamma-(S)-glutamylamino-N-hexylpentamide [Adang, Brussee, van der Gen and Mulder (1991) J. Biol.

Polymorphism in the glutathione conjugation activity of human erythrocytes towards ethylene dibromide and 1,2-epoxy-3-(p-nitrophenoxy)-propane.

In this study a polymorphism in the conjugating activity of human erythrocyte cytosol towards the dihaloethane, ethylene dibromide (EDB; 1,2-dibromoethane) was found. Two out of 12 human erythrocyte cytosols did not catalyze the formation of glutathione (GSH) conjugates of [1,2-14C]EDB. Ten cytosols formed the S,S'-ethylenebis(GSH) conjugate at a rate ranging from 0.

Active-site tyrosyl residues are targets in the irreversible inhibition of a class Mu glutathione transferase by 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone.

The mode of inactivation of glutathione S-transferase isoenzyme 3-3 from rat by the active site-directed inhibitor 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GSTCBQ) has been investigated by a combination of site-specific mutagenesis and mass spectrometric analysis of the sites of reaction of the reagent with the enzyme. This very reactive reagent is shown to target 3 residues in or near the active site, including the hydroxyl groups of Tyr-6 and Tyr-115 and the sulfhydryl group of Cys-114. Although the covalent attachment of one 2-(S-glutathionyl)dichloro-1,4-benzoquinonyl group/active site is sufficient to inactivate the enzyme ( < 5% residual activity), the 1 mol of reagent appears to be distributed among all three target sites.

Reversible conjugation of ethacrynic acid with glutathione and human glutathione S-transferase P1-1.

The reversibility of the conjugation reaction of the diuretic drug ethacrynic acid (EA), an alpha,beta-unsaturated ketone, with glutathione and glutathione S-transferase P1-1 (GST P1-1) has been studied. When the glutathione conjugate of EA was incubated with a 5-fold molar excess of N-acetyl-L-cysteine or GST P1-1, a time-dependent transfer of EA to N-acetyl-L-cysteine or GST P1-1 was observed. With increasing pH, the pseudo first order rate constants of transfer of EA to N-acetyl-L-cysteine increased from 0.

Inhibition of human glutathione S-transferases by dopamine, alpha-methyldopa and their 5-S-glutathionyl conjugates.

The reversible and irreversible inhibition of human glutathione S-transferases (GST) by dopamine, alpha-methyldopa and their 5-S-glutathionyl conjugates (termed 5-GSDA and 5-GSMDOPA, respectively) was studied using purified isoenzymes. The reversible inhibition, using CDNB as substrate and expressed as I50, ranged from 0.18-0.

The effect of a natural high-fibre diet on faecal and biliary bile acids, faecal pH and whole-gut transit time in man. A controlled study.

Dietary fibre possibly protects against colonic cancer by effects on bile acid metabolism. We investigated the effect of a natural high-fibre diet on secondary bile acid formation. Twelve healthy subjects on an habitual low-fibre diet (for 4 weeks) consumed a high-fibre menu for 10 weeks (experimental group).

Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases.

1. The diuretic drug ethacrynic acid (EA) is a potent reversible inhibitor of rat and human glutathione S-transferases (GST), with I50-values (microM) of 4.6-6.