Species variation in hepatic aldehyde oxidase activity

The activity of hepatic aldehyde oxidase from rabbit, guinea pig, rat, marmoset, dog, baboon and man was investigated in vitro with charged and uncharged N-heterocyclic substrates: Km and Vmax values were determined for phthalazine, 6,7-dimethoxy-1-[-4-(ethylcarbamoyloxy)piperidino]phthalazine (carbazeran), quinine and quinidine. The oxidation of N-phenylquinolinium chloride to N-phenyl-2-quinolone and N-phenyl-4-quinolone was followed spectrophotometrically. Rat or dog liver showed low and negligible enzyme activity respectively, whereas baboon liver contained a highly active aldehyde oxidase. Enzyme from marmoset and guinea pig liver had the closest spectrum of activity to human liver aldehyde oxidase. Unlike that from man, rabbit hepatic aldehyde oxidase was refractory towards carbazeran and converted N-phenylquinolinium chloride predominantly to the 2-quinolone. N-Phenyl-4-quinolone was the major oxidation product with enzyme from guinea pig, marmoset, baboon and man.     

Species differences in the toxicity and cytochrome P450 IIIA-dependent metabolism of digitoxin

In rats, cytochrome P450 (P450) IIIA enzymes are an important determinant of digitoxin toxicity. Induction of these liver microsomal enzymes decreases the toxicity of digitoxin by increasing its oxidative cleavage to digitoxigenin bis- and monodigitoxoside (dt2 and dt1). The present study shows that the susceptibility of different mammalian species to digitoxin toxicity is inversely related to liver microsomal P450 IIIA activity (measured as testosterone 6 beta-hydroxylase activity). Based on this correlation, we correctly predicted that hamsters, which have the highest P450 IIIA activity, are extremely resistant to digitoxin toxicity. To further examine the relationship between digitoxin toxicity and P450 IIIA activity, the pathways of digitoxin metabolism catalyzed by liver microsomes from nine mammalian species were examined by high performance liquid chromatography. The overall rate of digitoxin metabolism varied approximately 90-fold and followed the rank order: hamster greater than rat greater than guinea pig greater than dog greater than mouse approximately monkey greater than rabbit approximately cat greater than human. The qualitative differences in digitoxin metabolism were as striking as the quantitative differences. Formation of 16- and/or 17-hydroxydigitoxin was the major pathway of digitoxin oxidation catalyzed by liver microsomes from hamster, guinea pig, rabbit, cat, dog, and cynomolgus monkey. Guinea pig and, to a lesser extent, hamster liver microsomes also converted digitoxin to an unknown metabolite, the formation of which was catalyzed by P450. None of the species examined catalyzed the 12-hydroxylation of digitoxin to digoxin at a high rate. Similarly, none of the species examined catalyzed a high rate of conversion of digitoxin to dt2, with the notable exception of the rat. However, dt2 formation was the major pathway of digitoxin metabolism catalyzed by human liver microsomes, although humans were much less active (approximately 2%) than rats in this regard. The rate of dt2 formation varied approximately 41-fold among 22 samples of human liver microsomes, which was highly correlated (r = 0.841) with the rate of testosterone 6 beta-hydroxylation. Antibody against rat P450 IIIA1 inhibited the high rate of dt2 formation by rat liver microsomes and the low rate catalyzed by mouse, guinea pig, dog, monkey, and human liver microsomes. In contrast, anti-P450 IIIA1 did not inhibit the 12-, 16-, or 17-hydroxylation of digitoxin (or the formation of the unknown metabolite), despite the fact that anti-P450 IIIA1 strongly inhibited (greater than 70%) the 6 beta-hydroxylation of testosterone by liver microsomes from each of the species examined (except rabbit liver microsomes, which were inhibited only approximately 30%).(ABSTRACT TRUNCATED AT 400 WORDS)     

Deformylation of 4,4'-diformamidodiphenyl sulfone (DFD) by mammalian liver homogenates

Deformylation of 4,4'-diformamidodiphenyl sulfone (DFD) was studied in liver homogenates. The rates of deformylation were found to be in the following order: guinea pig ≧ human > mouse = rabbit = rat > dog.     

Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.     

Glucose-6-phosphate dehydrogenase and gama-glutamyltranspeptidase activities in the liver during chemically induced hepatocarcinogenesis in rats and mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) and γ-glutamyltranspeptidase (γ-GTP) were studied in the liver of rats and mice receiving a diet which contained 3′-methyl-4-(dimethylamino)azobenzene (3′-Me-DAB), diethylnitrosamine (DEN), or o-aminoazotoluene (o-AT). Elevated G-6-PDH activity was first observed within 2 weeks after the start of 3′-Me-DAB feeding, reaching a maximum at 4 weeks. There was a decrease from the 4-week level at about 6 weeks, followed by a sustained increase. The maintained activity after 8 weeks was about 6 times higher than in control rat liver. γ-GTP activity in rat liver increased immediately after the start of 3′-Me-DAB feeding; it was about 12 times the control value after 3–4 weeks. At 5–6 weeks, the activity decreased somewhat from the 3 to 4-week level, but this was followed by a further sustained increase. In rats receiving the DEN-containing water, the liver G-6-PDH and γ-GTP activities changed in the same way as in the 3′-Me-DAB-fed rats. Both activities increased in the early stage, reaching a maximum by 6 weeks, subsided at 7 weeks, and rose again from 9 weeks. In mice receiving o-AT-containing diet, hepatic G-6-PDH activity also changed in a biphasic pattern and closely related with that of liver γ-GTP activity.     

Investigations on the in vitro metabolism of five synthetic 19-norprogestins using hepatocyte suspensions isolated from five laboratory animal species

Five synthetic progestins of the 19-nortestosterone type (norethisterone, NET; levonorgestrel, LN; gestodene, GEST; NET-3-oxime, NETO; norgestimate, NGM) were investigated in the in vitro hepatocyte model. Radiolabelled progestins were added to hepatocyte suspensions (3 x 10(6) cells/ml) freshly prepared from female rat, guinea pig, rabbit, dog (beagle) and cynomolgus monkey. Drug level decreases (NET, LN, GEST) and prodrug conversions (NETO, NGM) were followed by radiochromatography (HPLC) for 60 min. In the case of NET and NETO the conversion into ethinyl estradiol (EE2) was quantified by RIA after HPLC separation. Half-lives of drug level decreases (t1/2), areas under the curves (AUC) and metabolic clearance rates (MCR) were estimated for all progestins. For NETO and NGM the percentages of conversion into NET and LN were calculated, respectively, and levels of EE2 determined in the case of NET and NETO. Rat hepatocytes showed an extremely high metabolic activity towards NET, LN and GEST resulting in t1/2 values of below 2 min. Respective values for rabbit hepatocytes ranged from 5-8 min, whereas half-lives calculated for liver cells from guinea pig, dog and monkey were generally above 30 min. A drastic increase in t1/2 was found for NETO (as compared to NET) in hepatocytes from rat, rabbit and monkey but not from guinea pig. Dog hepatocytes degraded NETO about 3 times more rapidly than NET. NGM was degraded much faster than LN in hepatocytes from all species except the rat. Liver cells from guinea pig and dog seem to be able to metabolize the 3-oxime group much more rapidly than hepatocytes from the other animal species. The lowest degree of prodrug conversion of 4% was observed for NGM and dog hepatocytes. Elevated EE2 levels were found in all experiments with NET and NETO. Results of NET, LN and GEST were compared with published in vivo experiments. No correlations were found for t1/2, MCR, and AUC.     

Species differences in the substrate specificity of hepatic cytochrome P-448 from polycyclic hydrocarbon-treated animals.

The in vitro effects of α-naphthoflavone on four hepatic mono-oxygenase activities associated with aromatic hydrocarbon responsiveness in the mouse (aryl hydrocarbon hydroxylase, 2-acetylamino-fluorene N-hydroxylase, biphenyl 2-hydroxylase, and biphenyl 4-hydroxylase) were investigated before and after methylcholanthrene treatment of C57BL/6N and DBA/2N mice, rats, hamsters, guinea pigs and rabbits. The electrophoretic pattern of cytochrome P-450 subunits and reduced CO-hemoprotein difference spectra of the microsomal fractions were also studied. Pretreatment of animals with methylcholanthrene caused: (1) a 1.5 to 2 mm hypsochromic shift in the Soret peak of the reduced hemoprotein-CO complexes in liver microsomes from a C57BL/6N mouse, rat, hamster and rabbit; a 0.5-nm hypsochromic shift in the guinea pig and no shift in the DBA/2N mouse; and (2) an increase in cytochrome P-450 apoproteins of the following molecular weights on sodium dodecyl sulfate-polyaerylamide gel electrophoresis: 54,000 and 55,000 in the C57BL/6N mouse; 48,000, 54,000 and 55,000 in the rat; 49,000 and 54,000 in the hamster; and 54,000 and 57,000 in the rabbit; a small increase in the 54,000 band was seen in the DBA/2N mouse and no increase in the guinea pig. In vitro addition of α-naphthoflavone selectively inhibited all four mono-oxygenase activities from the methylcholanthrene-treated C57BL/6N mouse, rat and hamster; 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the rabbit; and aryl hydrocarbon hydroxylase, 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the guinea pig. The addition of α-naphthoflavone enhanced the activities of aryl hydrocarbon hydroxylase and biphenyl 2-hydroxylase in liver microsomes from both control and methylcholanthrenetreated rabbits, but only biphenyl 2-hydroxylase activity was increased in the guinea pig: the activitity of 2-acetylaminofluorene N-hydroxylase was increased in both control and methylcholan-threne-treated DBA/2M mice, but only in the control C57BL/6N mouse. These data indicate that hepatic cytochrome P-448 is composed of multiple cytochromes, which differ among animal species, each catalyzing different mono-oxygenase activities.     

Characterization of non-peptide bradykinin B2 receptor agonist (FR 190997) and antagonist (FR 173657)

Pharmacologic parameters for a novel non-peptide bradykinin (BK)-B2 receptor agonist, 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoylcinnamidoacetyl]-N-+ ++methylano] benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline (FR 190997) (pEC50, ED50 values) and for the antagonist (E)-3-(6-acetamido-3-pyridyl)-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl ) oxymethyl] phenyl]-N-methylaminocarbonylmethyl] acrylamide (FR 173657) (pIC50, ID50 values) were measured using conventional contractile B2 receptor bioassays from rabbit, guinea pig and rat tissues and by mean of animal blood pressure models performed on anesthetized animals in the same species. In vitro assays (on the rabbit jugular vein and the guinea pig ileum) demonstrated that both the onset and duration of action of FR 190997 are prolonged compared to BK. These in vitro effects of FR 190997 strongly desensitized upon repeated tissue applications. Similar pEC50 values (7.7) were measured on the rabbit and the guinea pig tissues. In vivo, when injected intraarterially, FR 190997 produced hypotensive responses in rabbits and guinea pigs with ED50 values of 3.7 +/- 0.5 and 8.9 +/- 3.6 nmol/kg, respectively. Both the contractile and the hypotensive effects of FR 190997 were abolished by pretreating tissues (1 microM) or animals (0.1-0.5 micromol/kg) with D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]BK (HOE 140) or FR 173657. FR 173657 (pIC550 approximately 8.40), as well as other known antagonists (e.g., HOE 140, D-Arg-[Hyp3,D-Phe7,Leu8]BK), inhibited the in vitro myotropic effects of BK on the rabbit, guinea pig and rat tissues. FR 173657 also abrogated the in vivo hypotensive responses elicited by BK in the rabbit (ID50 57 +/- 9 nmol/kg), the guinea pig (ID50 215 +/- 56 nmol/kg) and the rat (ID50 187 +/- 50 nmol/kg). The in vivo duration of action of FR 173657 was significantly lower in the rabbit (= 20 min) than in the guinea pig and the rat (> 90 min). It is concluded that the non-peptides FR 190997 and FR 173657 enable efficient activation and antagonism of rabbit and guinea pig B2 receptors. These non-peptide molecules represent a marked progress in medicinal chemistry and may be useful to define the role played by the kallikrein/kinin system in vivo.     

Characterization of the purinoceptors present in rabbit and guinea pig liver

Micromolar concentrations of ATP induced a cAMP-independent glycogenolytic response in rabbit and guinea pig hepatocytes. With ATP alpha[35S] (adenosine 5'-[alpha-[35S]thio]triphosphate) as radioligand, we detected the presence of specific purinoceptors on hepatocytes and liver plasma membranes of both species. We determined a Kd value of 0.28 microM and a Bmax of 4.8 pmol/10(6) cells for rabbit hepatocytes and a Kd of 0.25 microM and a Bmax of 7.0 pmol/10(6) cells for guinea pig hepatocytes. The Kd values with purified plasma membranes from rabbit and guinea pig liver, were respectively 0.2 and 0.1 microM whereas the Bmax values were respectively 71 and 47 pmol/mg of protein. These purinoceptors belong to the P2Y-subclass as is evidenced by the high degree of similarity which exists between the binding affinities of several ATP analogues to either rabbit or guinea pig liver plasma membranes and rat liver plasma membranes, previously shown to possess P2Y-purinoceptors.     

Pharmacological action of cattle prostate extracts (PE). V. Effect on the bladder]

As the extract of cattle prostate (PE) is clinically effective in treating prostatic hypertrophy, a study was carried out on urinary bladders of rat, guinea pig, rabbit and dog as well as on guinea pig ileum. Muscle strip of rat and/or dog bladder contracted with PE with increasing spontaneous movement, and was unaffected by atropine. The isolated ileum of guinea pig also contracted with PE, and the contraction was inhibited by papaverine, but not by atropine. A rise in intravesical pressure was observed with increasing spontaneous movement in guinea pig bladder treated with PE, as well as in rabbit bladder in vitro or in situ. The sphincter vesica of guinea pig was dilated by PE as well as by ACh and methacholine.     

Animal models for percutaneous absorption

Animal models are important tools to predict human in vivo percutaneous absorption/penetration. Monkey, pig, rat, rabbit, guinea pig, hairless rodents, such as hairless rat, hairless mouse, hairless guinea pig and hairless dog, are among the most used animals for this purpose. Each animal model has its own advantages and weakness or limitation. To better correlate animal data with human skin absorption, we need to be familiar with each animal model's characteristics as well as experimental method and condition. We reviewed the original papers published after 1993 that described permeability of both animal skin and human skin. It showed that monkey, pig and hairless guinea pig are more predictive of human skin absorption/penetration and common laboratory animals, such as rat, rabbit, guinea pig, generally overestimate human skin absorption/penetration. Copyright © 2014 John Wiley & Sons, Ltd.     

Some Metabolites of S-Pentyl-L-cysteine in the Rabbit and Other Species

Abstract

1. The metabolism of S-pentyl-L-cysteine has been investigated in the guinea pig, hamster, mouse, rabbit and rat and in vitro by liver slices of these animals and of the pigeon and by kidney slices of mouse, rabbit and rat.

2. The amounts of pentylmercapturic acid, 3-(pentylthio)lactic acid and 3-(pentylthio)pyruvic acid excreted have been measured.

3. All the species examined excreted pentylmercapturic acid, the amount varying from 2% of the dose by the guinea pig to 73% by the hamster. 3-(Pentylthio)pyruvic and 3-(pentylthio)lactic acids were not detected in the urine of the hamster or rat but were excreted by the other species in amounts approximately the same as those of pentylmercapturic acid.

4. ‘Hydroxypentylmercapturic’ acids were excreted by mouse, rabbit and rat and were identified in the case of the rabbit and rat.

5. 4-Carboxybutylmercapturic acid was identified in the urine of dosed mice, rabbits and rats. A further metabolite in rat urine was tentatively identified as 3-(4-carboxybutylthio)lactic acid.

6. Pentylmercapturic acid sulphoxide was detected in the urine of dosed guinea pigs and mice.

7. All tissue preparations examined converted pentyl-L-cysteine to pentylmercapturic acid. 3-(Pentylthio)lactic acid and 3-(pentylthio)pyruvic acid were formed by all tissues examined although only in trace amounts by hamster liver and rat kidney slices. All tissues examined formed hydroxymercapturic acids' although only traces were detected in digests containing hamster liver slices. 4-Carboxybutylmercapturic acid was formed in rabbit liver digests. With rabbit kidney a metabolite thought to be 3-(4-carboxybutylthio)lactic acid was produced.     

Serotonin (5-hydroxytryptamine) glucuronidation in vitro: assay development,human liver microsome activities and species differences

1. The main purpose was to develop a high-performance liquid chromatography (HPLC)-based method to assay serotonin glucuronidation activity using liver microsomal fractions. Application of this method was then demonstrated by determining serotonin UDP-glucuronosyltransferase (UGT) enzyme kinetics using human liver microsomes and recombinant human UGT1A6. Interspecies differences were also evaluated using liver microsomes from 10 different mammalian species. 2. Incubation of liver microsomes with serotonin, UDP-glucuronic acid and magnesium resulted in the formation of a single product peak using HPLC with fluorescence and ultraviolet absorbance detection. This peak was confirmed as serotonin glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 352 with positive-ion mass spectrometry. 3. Following a preparative HPLC isolation, the structure of this metabolite was established as serotonin-5-O-glucuronide by (1)H-NMR spectroscopy. 4. Enzyme kinetic studies showed apparent K(m) and V(max) of 8.8 +/- 0.3 mM and 43.4 +/- 0.4 nmoles min(-1) mg(-1) protein, respectively, for human liver microsomes, and 5.9 +/- 0.2 mM and 15.8 +/- 0.2 nmoles min(-1) mg(-1), respectively, for recombinant UGT1A6. 5. The order of serotonin-UGT activities in animal liver microsomes was rat > mouse > human > cow > pig > horse > dog > rabbit > monkey > ferret. Cat livers showed no serotonin-UGT activity. Heterozygous and homozygous mutant Gunn rat livers had 40 and 13%, respectively, of the activity of the normal Wistar rat, indicating a significant contribution by a rat UGT1A isoform to serotonin glucuronidation. 6. This assay provides a novel sensitive and specific technique for the measurement of serotonin-UGT activity in vitro.     

Species Differnces in the Hydrolysis of Meperdine and its Inhibition by Organophosphate Compounds

Species Differences in the Hydrolysis of Meperidine and ItsInhibition by Organophosphate Compounds. Luttrell, W. E., andCastle, M. C. (1988). Fundam. Appl. Toxicol. 11, 323–332.The hydrolysis of meperidine was assayed in washed, unfortifiedliver microsomal fractions of guinea pig, rat, mouse, dog, andhuman, by following substrate disappearance as quantitated byhigh-performance liquid chromatography. Using the method ofLineweaver-Burk plots, the velocity of the meperidine hydrolysisreaction was not detectable in guinea pig, very low in human,and extremely high in dog. Hydrolysis of p-nitrophenyl acetatewas also monitored in liver microsomal preparations from thesame animal species, with guinea pig showing greatest hydrolyticactivity and rat showing least hydrolytic activity for thissubstrate. The data in the above two assays suggested that meperidinehydrolysis is mediated by a unique esterase not present in guineapig and very low in human, but present with high activity indog liver micro-somes. From these comparative studies we concludedthat liver microsomes from different species may contain differentcarboxylesterases having different affinities for meperidine.To further characterize meperidine carboxylesterase of dog andrat liver microsomes, inhibitory studies in vitro with two organophosphatecompounds—paraoxon (diethyl-p-nitrophenyl phosphate) andsoman (pinacolyl methylphosphonofluoridate)—indicateda varied pattern of enzyme inhibition. These results suggestedthat liver microsomal carboxylesterases are involved in themetabolism of meperidine and that interference with these enzymesby organophosphate compounds may alter pharmacologic and toxicologiceffects of meperidine.     

Neuropeptide Y1- and Y2-receptor-mediated cardiovascular effects in the anesthetized guinea pig, rat, and rabbit

Neuropeptide Y (NPY) causes vasoconstriction through Y1-receptors and inhibits vagal bradycardia through presynaptic Y2-receptors. These effects of NPY were investigated in anesthetized guinea pigs, rats, and rabbits to find the most suitable species for evaluation of Y1- and Y2-active agents in vivo. The increase in blood pressure (through Y1) of lower doses of NPY was similar in the three species (ED50, 0.9 +/- 0.13, 0.8 +/- 0.39, and 0.6 +/- 0.09 nmol/kg, respectively), but higher doses had depressor effects in four of six rats. Vagal bradycardia, induced by electrical stimulation of the right cervical vagus nerve, was inhibited by NPY in the guinea pig and in the rat (ED35, 3.5 +/- 0.46 and 11.2 +/- 1.79 nmol/kg, respectively; p < 0.05) but not in the rabbit. In the guinea pig, the Y2-receptor-preferring fragment NPY(3-36) and the selective Y1-receptor antagonist H 409/22 were used to confirm that the increase in blood pressure was mediated solely through the Y1-receptor and the vagal inhibition solely through the Y2-receptor. Aside from the cardiovascular effects, NPY caused a decrease in the body temperature and inhibited vagal bronchoconstriction in this species. Considering that NPY may cause depressor effects in the rat and has no effect on the vagal bradycardia in the rabbit, the guinea pig is preferable to both these species for assessment of Y1- and Y2-receptor-active agents in vivo.     

Oxymorphone metabolism and urinary excretion in human, rat, guinea pig, rabbit, and dog

Oxymorphone was extensively metabolized by human, rat, dog, and guinea pig and to a lesser extent by rabbit. The most abundant metabolite in urine for all species was conjugated oxymorphone (12.7-81.7% administered dose) followed by 6 beta- and 6 alpha-carbinols produced by 6-keto reduction of oxymorphone. 6 beta-Oxymorphol (0.2-3.1%) was found in the urine of all species, whereas 6 alpha-oxymorphol (0.1-2.8%) was found only in human, rabbit, and guinea pig. Small amounts of free oxymorphone (less than or equal to 10%) were excreted by all species except rabbit, which excreted 31.7%. Overall recoveries of oxymorphone and metabolites from urine ranged from 15-96%, of which greater than 80% was excreted in the first 24 hr by all species except dog. Only 35% was excreted by dog during the first day. Stereoselectivity of 6-keto- reduction was observed for all species with the 6 beta-carbinol metabolite being most abundant in the urine of all but guinea pig. Considerable individual variability occurred in the excretion of free and conjugated oxymorphone by six human subjects following oral dosing. Species trends in the metabolism of 6-keto-opioids are discussed.     

In vitro metabolism of simazine,atrazine and propazine by hepatic cytochrome P450 enzymes of rat,mouse and guinea pig,and oestrogenic activity of chlorotriazines and their main metabolites

1. The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ) and propazine (PRZ) in liver microsomes from rat, mouse and guinea pig and the oestrogenic activity of chlorotriazines and their main metabolites have been studied. 2. The formation rates of products in chlorotriazine metabolism were determined by HPLC. The principal reactions catalysed by the cytochrome P450 (P450) system were N- monodealkylation and isopropylhydroxylation in all liver microsomes. As a result, 2- chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), and 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5- triazine (M3)(ATZ-M3for ATZ) and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N- bidealkylation was not found in this system. 3. The formation rates of N-deethylated metabolites (SIZ-M1 and ATZ-M2) were generally higher in mouse than in rat and guinea pig. The formation rates of N- deisopropylated metabolites (ATZ-M1 and PRZ-M2) in guinea pig were the lowest among the three animal species. The formation rates of isopropylhydroxylated metabolites (ATZM3 and PRZ-M4) were remarkably low in mouse compared with rat and guinea pig. 4. The enzyme kinetics of chlorotriazine metabolism were examined by Eadie-Hofstee analyses. Some species differences in Michaelis-Menten parameters for each metabolite were observed, and the ranking orders were varied among the metabolites. 5. The binding affinity of chlorotriazines (SIZ, ATZ and PRZ) and their metabolites (M1-4) for recombinant human oestrogen receptor-alpha was assayed using the fluorescence polarization method. The binding a nity of M2 was significantly higher than those of parent compounds and other metabolites, although the oestrogenic activity was remarkably low compared with that of 17 beta-oestradiol (E2). 6. These results suggest that the pattern of metabolism of SIZ, ATZ and PRZ by the P450 system differs extensively among rat, mouse and guinea pig, and that M2 may be an activated metabolite of chlorotriazines.     

In vitro metabolism of simazine, atrazine and propazine by hepatic cytochrome P450 enzymes of rat, mouse and guinea pig, and oestrogenic activity of chlorotriazines and their main metabolites

1. The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ) and propazine (PRZ) in liver microsomes from rat, mouse and guinea pig and the oestrogenic activity of chlorotriazines and their main metabolites have been studied. 2. The formation rates of products in chlorotriazine metabolism were determined by HPLC. The principal reactions catalysed by the cytochrome P450 (P450) system were N-monodealkylation and isopropylhydroxylation in all liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), and 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ) and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazi ne (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N-bidealkylation was not found in this system. 3. The formation rates of N-deethylated metabolites (SIZ-M1 and ATZ-M2) were generally higher in mouse than in rat and guinea pig. The formation rates of N-deisopropylated metabolites (ATZ-M1 and PRZ-M2) in guinea pig were the lowest among the three animal species. The formation rates of isopropylhydroxylated metabolites (ATZ-M3 and PRZ-M4) were remarkably low in mouse compared with rat and guinea pig. 4. The enzyme kinetics of chlorotriazine metabolism were examined by Eadie-Hofstee analyses. Some species differences in Michaelis-Menten parameters for each metabolite were observed, and the ranking orders were varied among the metabolites. 5. The binding affinity of chlorotriazines (SIZ, ATZ and PRZ) and their metabolites (M1-4) for recombinant human oestrogen receptor-alpha was assayed using the fluorescence polarization method. The binding affinity of M2 was significantly higher than those of parent compounds and other metabolites, although the oestrogenic activity was remarkably low compared with that of 17beta-oestradiol (E2). 6. These results suggest that the pattern of metabolism of SIZ, ATZ and PRZ by the P450 system differs extensively among rat, mouse and guinea pig, and that M2 may be an activated metabolite of chlorotriazines.     

Species differences in the hydroxylation of aniline and N-ethylaniline by liver microsomes

Summary The velocity of the N- and p-hydroxylation of aniline and N-ethylaniline by NADPH-dependent hydroxylases in guinea pig liver microsomes was found to be as high as in rabbit liver microsomes. Microsomes prepared from cat livers were less active.The effect of 2,4-dichlorophenol, p-chloromercuribenzoate, semicarbazide, and 8-hydroxyquinoline on the microsomal hydroxylations was observed to be not uniform in the microsomes of the species studied.Carbon monoxide inhibited the p-hydroxylation of N-ethylaniline by the microsomes prepared from the livers of rabbits, guinea pigs, dogs, and rats. The N-hydroxylation of N-ethylaniline by microsomes from rat's liver was inhibited like the p-hydroxylation by carbon monoxide. The N-hydroxylation by dog and guinea pig microsomes was scarcely, if at all, inhibited by the same carbon monoxide pressure. These enzymes showed a lower affinity for oxygen than the N-hydroxylating enzyme in rat microsomes and the p-hydroxylating enzymes.With 1 Figure in the TextResults of this study were presented at a meeting of the Deutsche Pharmakologische Gesellschaft in Mainz on April 26, 1965.     

Hydrolysis of 3H-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro

Tritiated bambuterol, a bis-dimethylcarbamate prodrug of terbutaline, was incubated in vitro with blood from both sexes of the following species: man, guinea pig, rat, mouse, dog and rabbit. The rates of hydrolysis of bambuterol to its monocarbamate derivative and further to terbutaline were measured. Large species variations were observed, e.g. blood from two of the human subjects was 15-fold more active than blood from the male rats. The rate of terbutaline formation as a function of initial bambuterol concentration was investigated in human plasma, and was found to describe a bell-shaped curve. Several pieces of evidence indicated that butyrylcholinesterase (EC 3.1.1.8) is the blood enzyme predominantly responsible for hydrolysis of bambuterol, although minor contributions from other esterases cannot be excluded. An exception may be blood from the rabbit, where the kinetics of the hydrolysis was different than in blood from the other species. The kinetics of bambuterol hydrolysis is discussed on basis of the established mechanism of carbamate interactions with cholinesterases, and the high affinity of bambuterol for butyrylcholinesterase.