Studies on the role of folic acid and folate-dependent enzymes in human methanol poisoning

Methanol toxicity is observed in monkeys and humans but is not seen in rats or mice. The expression of methanol poisoning is related to the ability of an animal to metabolize formate to carbon dioxide. Since the rate of formate oxidation is related to hepatic tetrahydrofolate (H4folate) content and the activities of folate-dependent enzymes, studies were designed to determine hepatic concentrations of H4folate and activities of folate-dependent enzymes of human liver and livers of species considered insensitive to methanol poisoning. An excellent correlation between hepatic H4folate and maximal rates of formate oxidation has been observed. In human liver, H4folate levels were only 50% of those observed for rat liver and similar to those found in monkey liver. Total folate was also lower (60% decreased) in human liver than that found in rat or monkey liver. Interestingly, mouse liver contains much higher hepatic H4folate and total folate than rat or monkey liver. This is consistent with higher formate oxidation rates in this species. A second important observation has been made. 10-Formyltetrahydrofolate dehydrogenase activity, the enzyme catalyzing the final step of formate oxidation to carbon dioxide, was markedly reduced in both monkey and human liver. Thus, two mechanisms may be operative in explaining low formate oxidation in species susceptible to methanol toxicity, low hepatic H4folate levels and reduced hepatic 10-formyltetrahydrofolate dehydrogenase activity.     

Developmental expression of aldehyde dehydrogenase in rat: a comparison of liver and lung development.

Metabolism is one of the major determinants for age-related changes in susceptibility to chemicals. Aldehydes are highly reactive molecules present in the environment that also can be produced during biotransformation of xenobiotics and endogenous metabolism. Although the lung is a major target for aldehyde toxicity, early development of aldehyde dehydrogenases (ALDHs) in lung has been poorly studied. The expression of ALDH in liver and lung across ages (postnatal day 1, 8, 22, and 60) was investigated in Wistar-Han rats. In adult, the majority of hepatic ALDH activity was found in mitochondria, while cytosolic ALDH activity was the highest contributor in lung. Total aldehyde oxidation capability in liver increases with age, but stays constant in lung. These overall developmental profiles of ALDH expression in a tissue appear to be determined by the different composition of ALDH isoforms within the tissue and their independent temporal and tissue-specific development. ALDH2 showed the most notable tissue-specific development. Hepatic ALDH2 was increased with age, while the pulmonary form did not. ALDH1 was at its maximum value at postnatal day 1 (PND1) and decreased thereafter both in liver and lung. ALDH3 increased with age in liver and lung, although ALDH3A1 was only detectible in lung. Collectively, the present study indicates that, in the case of aldehyde exposure, the in vivo responses would be tissue and age dependent.     

Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: comparison with dihydrocoumarin and other coumarin metabolites

Single doses of coumarin (125 mg/kg, ip) produced a depletion of hepatic nonprotein sulfhydryl groups (mainly reduced glutathione; GSH) in young male Sprague-Dawley rats after 2 hr and increased liver weight and produced hepatic centrilobular necrosis after 24 hr. Coumarin also produced time- and dose-dependent toxic effects in primary rat hepatocyte cultures. A marked reduction of GSH levels was also observed in vitro and this was not due either to the formation of oxidized glutathione (GSSG) or to the leakage of GSH and/or GSSG from the hepatocytes. Coumarin-induced toxicity in rat hepatocytes could be inhibited by the cytochrome P450 inhibitors ellipticine and metyrapone and potentiated by depleting hepatocyte GSH levels with diethyl maleate. In contrast to coumarin, dihydrocoumarin--which lacks the 3,4-double bond--produced little toxicity in rat hepatocytes either in vivo (127 and 254 mg/kg, ip) or in vitro. Similarly, coumarin was more toxic to rat hepatocytes than a number of known coumarin metabolites including 3- and 7-hydroxycoumarin and o-hydroxyphenylacetic acid. The results of these studies demonstrate a good in vivo/in vitro correlation for the effects of coumarin and dihydrocoumarin in rat hepatocytes. Furthermore, the data suggest that coumarin hepatoxicity in the rat is due to coumarin bioactivation by cytochrome P450-dependent enzymes to a toxic metabolite(s), which may be a coumarin 3,4-epoxide intermediate. GSH appears to protect against coumarin-induced toxicity possibly by the formation of conjugates with the toxic coumarin metabolite(s).     

Inactivation mechanism of low-KM rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro.

The inactivation of low-KM rat liver mitochondrial aldehyde dehydrogenase (ALDH) by the alcohol-sensitizing agent cyanamide (H2NCN) has been studied in vitro. The effect of the concentrations of NAD+ at different concentrations of catalase on the inactivation of ALDH by cyanamide (20 and 200 microM) in vitro point to an ALDH-NAD(+)-catalase complex prior to the binding to cyanamide to form the holoenzyme-inhibitor complex. Cyanamide itself could be responsible for the inactivation of ALDH. The possibility that both irreversibly inactivated ALDH and cyanamide remain free at the end of the inactivation process is discussed. The effects of pH and ionic strength on the inactivation process are also described. The pseudo-first order rate constants for inactivation of low-KM ALDH depends on both effects, suggesting that electrostatic forces are involved in the process and that a group with pK approximately 6.8, presumably a histidine residue, at the active site of ALDH could be involved. A representative equation for the inactivation process of low-KM ALDH by cyanamide in vitro has been fitted to experimental kinetic data, involving both catalase and inhibitor concentrations.     

Effect of coenzyme Q10 on warfarin hydroxylation in rat and human liver microsomes

Our previous animal study has suggested that the accelerated metabolism of warfarin enantiomers with concurrent coenzyme Q(10) (CoQ(10)) treatment accounts for the reduced anticoagulant effect of warfarin in rats. The present study was to assess the effect of CoQ(310) on individual hydroxylation pathways of the in vitro microsomal metabolism of warfarin enantiomers and to extrapolate in vitro data to in vivo situation. The effect of the antioxidant CoQ(10) on the hydroxylation of warfarin enantiomers was examined using rat and human liver microsomes. Based on the in vitro kinetic data, together with the information retrieved from the literature, the magnitude of warfarin-CoQ(10) interaction in man was quantitatively predicted. In rat liver microsomes, CoQ(10) exhibited a selective activation effect on the 4'-hydroxylation of S-warfarin, with a K(A) value (i.e. dissociation constant of the enzyme-activator complex) being one third and one fifth of those for the 6- and 7-hydroxylation, respectively. The activation effect of CoQ(10) was selective towards the 6- and 7-hydroxylation of R-warfarin at low substrate concentrations, but towards the 4'-hydoxylation of the R-enantiomer at high substrate concentrations. In human liver microsomes, CoQ(10) was a selective activator of the 7-hydroxylation of both R- and S-enantiomers of warfarin, with K(A) values being half to one twelfth of those for the other pathways. A relatively accurate prediction was made for the increase in the total and hepatic clearance of both S- and R-warfarin in rats with concurrent CoQ(10) treatment based on their respective overall hydroxylation, when the active transport of CoQ(10)into the hepatocytes was taken into consideration. In man, one would expect about 32% and 17% increase in the total clearance of S- and R-warfarin, respectively, with coadministration of 100 mg CoQ(10). In both species, CoQ(10) had enzyme activation effect, which appeared to be regioselective but not stereoselective, on the formation of the phenolic metabolites of warfarin enantiomers. A moderate increase in the total clearance of warfarin enantiomers could occur with coadministration of CoQ(10)in humans.     

Isolation and purification of rat liver morphine UDP-glucuronosyltransferase

A UDP-glucuronosyltransferase (UDPGT) isoenzyme capable of morphine glucuronidation has been purified to apparent homogeneity and partially characterized from hepatic microsomes of female Wistar rats which have low 3 alpha-hydroxysteroid UDPGT. A rapid and sensitive assay was developed to quantify morphine glucuronide formation using 14C-UDP-glucuronic acid and reverse phase C-18 minicolumns whereby radioactive glucuronides were differentially eluted from 14C-UDP-glucuronic acid. Trisacryl-DEAE and chromatofocusing chromatographic procedures were employed to separate and purify morphine UDPGT in the presence of exogenous phosphatidylcholine. The addition of phospholipid was necessary to stabilize UDPGT activities throughout the purification procedures. Morphine UDPGT was isolated to apparent homogeneity and displayed a pl of 7.9 upon chromatofocusing. A monomeric molecular weight of 56,000 was obtained. The purified enzyme reacted with morphine but not with 4-hydroxybiphenyl, p-nitrophenol, testosterone, androsterone, estrone, bilirubin, 4-aminobiphenyl, or alpha-naphthylamine. The MgCl2 requirement for maximal expression of morphine glucuronidation was higher for the purified enzyme than for solubilized and intact microsomes. Codeine competitively inhibits morphine glucuronidation with an apparent Ki of 1.1 mM with the purified morphine UDPGT. 4-Hydroxybiphenyl UDPGT was separated from morphine UDPGT using a chromatofocusing procedure for Emulgen 911-solubilized microsomes. An apparent pl value of 5.5 was obtained for this protein. Based on this work we conclude that morphine and 4-hydroxybiphenyl can react with separate UDPGT isoforms.     

Effect of castration on the turnover of rat liver alcohol dehydrogenase

Castration increased liver alcohol dehydrogenase activity and enzyme protein in male rats. The turnover of alcohol dehydrogenase determined from the decline in radioactivity present in immunoprecipitated enzyme after injection of NaH¹⁴CO₃ was decreased after castration. The fractional rate of degradation (K_d) for the enzyme was 0.11 · day⁻¹ in the castrated as compared with 0.13 · day⁻¹ in the control animals (P < 0.05). The fractional rate of synthesis (K_s) of the enzyme was not affected by castration, while the absolute rate of synthesis was increased slightly. This study shows that a decrease in the rate of degradation is the principal cause for the increase in liver alcohol dehydrogenase following castration.     

Glucuronidation of the enantiomers of E-10-hydroxynortriptyline in human and rat liver microsomes

Conjugation of racemic E-10-hydroxynortriptyline (E-10-OH-NT) with glucuronic acid was studied in the liver microsomal fraction of rats and humans. The diastereomeric glucuronides of E-10-OH-NT were resolved and quantitated by HPLC. Only the (+)-enantiomer was glucuronidated in liver microsomes from humans. Rat liver microsomes catalyzed the formation of both glucuronides. Phenobarbital pretreatment of rats increased the glucuronidation of both enantiomers about five-fold. The formation rate of (+)-E-10-OH-NT glucuronide varied from 5.5 to 33.2 pmol/mg x min, in microsomes from 13 humans. High activity was found in individuals previously treated with pentobarbital. Inhibition experiments with human liver microsomes showed that amitriptyline is a potent competitive inhibitor of (+)-E-10-OH-NT glucuronidation. p-Nitrophenol, paracetamol and 2-hydroxydesipramine also inhibited this reaction.     

Differences in kinetic characteristics and in sensitivity to inhibitors between human and rat liver alcohol dehydrogenase and aldehyde dehydrogenase

1. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that liver alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected in both human and rat liver. The human isoenzymes corresponded to the ALDH-I and ALDH-II type. 3. In the rat, one isoenzyme had low Km and showed similar activity than in human liver but differed in their sensitivity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.     

Studies on the mechanism of action of benzamide riboside: a novel inhibitor of IMP dehydrogenase

Benzamide is a well known inhibitor of poly(ADP-ribose)polymerase, an enzyme involved in DNA repair. However, benzamide exhibited neuotoxicity in animals and hence, in the hope of overcoming this problem, benzamide riboside (BR) was synthesized. Our mechanism of action studies on BR suggested that the agent was being metabolized to its 5'-monophosphate and then to its NAD analogue (BAD, benzamide adenine dinucleotide) that inhibits Inosine 5'-monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme of the branched purine nucleotide synthetic pathway that provides guanylates including GTP and dGTP. There are two isoforms of IMPDH, type I that is constitutively present in all cells, and type II that is inducible and is present in highly proliferating cells such as cancer. Ongoing studies with BR analogues suggest that they are more selective in inhibiting IMPDH type II. Our studies have characterized the metabolites of BR, especially its NAD analogue, BAD, by synthesizing this active metabolite by enzymatic means, and identifying its structure by NMR and mass spectrometry. We have partially purified IMPDH from tumor cells and have examined the kinetics of inhibition of IMPDH by BAD. We have also compared biochemical and cytotoxic activities of BR with tiazofurin and selenazofurin, that share similar mechanisms of action with BR. Our studies demonstrated that 2-3-fold more BAD is formed compared to TAD and SAD, the active metabolites of tiazofurin and selenazofurin, respectively. BR has demonstrated potent cytotoxic activity in a diverse group of human tumor cells, specifically more active in sarcomas and CNS neoplasms compared to tiazofurin or selenazofurin. Future in vivo animal studies should set a stage for determining its effectiveness in clinical Phase I studies.     

Studies on the mechanism of clonidine-induced mydriasis in the rat

Intravenous administration of clonidine hydrochloride (3-100 micrograms/kg) produced a dose-dependent pupillary dilation in anaesthetized rats. All experiments were carried out in rats in which vagosympathetic nerve trunks were sectioned bilaterally at the cervical level. Clonidine-induced mydriasis was present only in those preparations having intact parasympathetic neural tone to the iris. Depletion of CNS monoamines by more than 95% with reserpine (5 mg/kg) and alpha-methyl-para-tyrosine (2 X 300 mg/kg) failed to alter the dose-response relation to clonidine. Pretreatment with the alpha-2-adrenoceptor antagonist, yohimbine hydrochloride (1.5 mg/kg), produced about a 10-fold shift to the right in the pupillary dose-response curve to clonidine. Yohimbine administered after the highest dose of clonidine also antagonized the mydriatic response. The above results suggest that clonidine acts on CNS post-synaptic alpha-2-adrenoceptors to produce mydriasis by withdrawal of parasympathetic neural tone to the iris. In an attempt to assess the physiological substrate(s) involved, mydriatic responses, due to parasympatho-inhibition, were evoked by electrical stimulation of ascending (sciatic nerve and medullary) and descending (hypothalamic) pathways. Yohimbine (0.3 and 1.0 mg/kg) produced a dose-dependent inhibition of the pupillary dilation evoked by stimulation of the sciatic nerve and medullary loci, whereas these doses of yohimbine failed to alter the dilation in response to hypothalamic stimulation. Similarly, monoamine depletion greatly antagonized the pupillary dilation elicited by sciatic nerve and medullary stimulation without significantly affecting mydriasis due to hypothalamic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and purification of two human liver UDP-glucuronosyltransferases

Two UDP-glucuronosyltransferases (EC 2.4.1.17) were purified from human liver microsomes. Human liver microsomes were solubilized with Emulgen 911 and the UDP-glucuronosyltransferases were separated and purified by chromatofocusing and UDP-hexanolamine Sepharose 4B affinity chromatography. One isoenzyme eluted with an apparent pl of 7.4, displayed a subunit molecular weight of 53,000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and catalyzed the glucuronidation of p-nitrophenol, 4-methylumbelliferone, alpha-naphthylamine, and estriol, but not that of 4-aminobiphenyl. A second isoenzyme eluted with an apparent pl of 6.2, displayed a subunit molecular weight of 54,000 after SDS-PAGE, and catalyzed the glucuronidation of p-nitrophenol, 4-methylumbelliferone, alpha-naphthylamine, and 4-aminobiphenyl, but not that of estriol. Neither of the purified human liver UDP-glucuronosyltransferases employed estrone, beta-estradiol, testosterone, androsterone, or 5 alpha-androstane-3 alpha,17 beta-diol as substrate. These enzymes displayed apparent Km values in the same order of magnitude for a given substrate. In general, high concentrations of phosphatidylcholine were required for reconstitution of maximal glucuronidation activity. This report documents the existence of multiple UDP-glucuronosyltransferases in human liver.     

Studies on the mechanism of the protective and antidotal actions of diazepam in organophosphate poisoning.

The effect of diazepam on soman-induced bradycardia and respiratory depression in rabbits has been investigated. Diazepam prevents the bradycardia produced by soman in conscious rabbits. Although atropine will reverse the respiratory depression produced by soman in anesthetized rabbits, diazepam enhances the depression and renders rabbits less susceptible to this action of atropine.