Role of metabolism by intestinal bacteria in arbutin-induced toxicity in vitro

A possible role of metabolism by intestinal bacteria in arbutin-induced toxicity was investigated in mammalian cell cultures. Following an incubation of arbutin with intestinal bacteria, either Bifidobacterium longum HY81 or Bifidobacterium adolescentis, for 24 h, its aglycone hydroquinone could be produced and detected in the bacterial culture media. The bacterial growth was not affected up to 10 mM arbutin in the culture medium. When the toxicity of bacteria cultured medium with arbutin was tested in the HepG2 cell lines, the medium with arbutin was more toxic than either parent arbutin only or bacteria cultured medium without arbutin, indicating that metabolic activation might be required in arbutin-induced toxicity. In addition, bacteria cultured medium with arbutin could suppress LPS and ConA mitogenicity in splenocyte cultures prepared from normal mice. The results indicate that the present toxicity testing system might be applied for assessing the possible role of metabolism by intestinal bacteria in certain chemical-induced toxicity in mammalian cell cultures.     

Effect of variations in acute and chronic iodine intake on the accumulation and metabolism of [35S]methimazole by the rat thyroid gland: Differences from [35S]propylthiouracil

Studies were performed to ascertain the effect of various levels of chronic iodine intake and varying doses of iodide [0.002–100 μmoles KI/100 g body weight (BW)] given acutely on the rat thyroid metabolism of [³⁵S]methimazole ([³⁵S]MMI, 8.76 μmoles/kg BW). Variations in both acute and chronic iodine intake were associated with as much as four-fold changes in thyroid levels of total ³⁵S and unchanged [³⁵S]MMI. Chronic low iodine intake resulted in a considerable reduction in the thyroid uptake of [³⁵S]MMI (40% decrease) from high or normal chronic iodine intake. Unlike [³⁵S]PTU studies, the effect of increasing acute iodide dosage produced a biphasic response in the thyroid uptake of [³⁵S]MMI only in low chronic iodine intake. In these animals 0.1 μmoles KI/100 g BW produced the maximum uptake of [³⁵S]MMI (300% increase) but had no effect on high or normal chronic iodine intake. In these latter groups of rats, thyroidal total ³⁵S increased to plateau levels with increasing acute iodide dosage in the range of 0.1–1 μmoles/100 g BW which were unaffected by increased iodide up to 100 μmoles/100 g BW. In low chronic iodine intake rats also, the thyroid ³⁵S level seen at 1 μmole/100 g was unaffected by increased iodide dosage up to 100 μmoles/100 g. The steady thyroid ³⁵S levels seen in this acute iodide dose range in low, normal and high chronic iodine rats were 100, 70 and 110% respectively greater than their control values. Unlike [³⁵S]PTU studies, in general, an increase in thyroid total ³⁵S achieved by varying acute or chronic iodine intake was found to be associated with a large increase in the percentage thyroid ³⁵S occurring as free inorganic sulphate with a consequent effect on thyroid unchanged [³⁵S]MMI. In chronic low iodine intake animals treated with acute radioidide, in agreement with [³⁵S]PTU studies, no direct correlation was found between thyroid uptake or oxidation of [³⁵S]MMI and thyroidal total iodine, the accumulation or organification of acute [¹²⁵I]iodide, the occurrence of the Wolff-Chaikoff effect or saturation of thyroid iodide transport.     

The uptake of [35S]methimazole by sheep thyroid slices in vitro

The uptake of [³⁵S]methimazole by sheep thyroid slices has been shown to be activated in the presence of iodide. The total uptake (Q) of [³⁵S]methimazole was shown to be the sum of a saturable process and a non-saturable process. The constants Q_max, K_s and P in the two-term equation were determined using a published statistical method and a Fortran IV computer programme. Diiodotyrosine (DIT) at a 0.1 mM concentration stimulated the saturable uptake of [³⁵S]methimazole appreciably in the absence of iodide, whilst thyroid-stimulating hormone (TSH) inhibited uptake in the presence of iodide and was of no effect in the absence of iodide. Propylthiouracil (PTU) inhibited the saturable uptake of [³⁵S]methimazole whilst perchlorate had no effect.     

In vitro metabolism of clindamycin in human liver and intestinal microsomes.

Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the S-oxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6beta-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (approximately 26%) by coincubation with clindamycin (100 microM). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.     

Biosynthesis of griseolic acids: incorporation of 13C-labeled compounds into griseolic acid A.

Biosynthesis of griseolic acids, competitive inhibitors of cyclic nucleotide phosphodiesterase, was investigated with the culture of a producing strain of Streptomyces griseoaurantiacus. 13C-Labeled and 15N-labeled compounds were added into the culture, and 13C-enriched and 15N-enriched griseolic acid A was isolated from the culture medium and analyzed by 13C NMR and 15N NMR spectroscopy. The compounds added to growth medium were [2-13C]acetate, [1,2-13C]acetate, [1,4-13C]succinate, [1-13C]glucose, [6-13C]glucose, [2-13C]ribose, and [1-13C, 15N]glycine. The results suggest that adenosine, which is formed from amino acids and sugars contributes the adenine and ribose moieties to griseolic acid A. The data also suggest that a dicarboxylic acid from the Krebs tricarboxylic acid cycle contributes to the dicarboxylic part of the compound.     

Alterations in [3H]thymidine incorporation into DNA and [3H]uridine incorporation into RNA induced by 5-azacytidine in vivo.

Administration in vivo of 5-azacytidine (5-aza-CR) caused suppression of [³H]thymidine ([³H]TdR) incorporation into DNA of bone marrow and gastrointestinal mucosa of mice and a more prolonged suppression of L1210 ascites tumor. Single doses of 5-aza-CR caused a modest and short-lived suppression of incorporation of [³H]uridine ([³H]UR) into nuclear RNA of L1210 ascites tumor cells. No suppression of [³H]UR incorporation into RNA of bone marrow or gastrointestinal mucosa was observed. L1210 tumor cells resistant to the other active cytidine analogue, cytosine arabinoside, demonstrated less disruption of [³H]TdR incorporation after exposure to 5-aza-CR, suggesting some cross resistance in the effects of these two drugs on DNA synthesis. Survival studies carried out in mice bearing both the sensitive and resistant L1210 tumor cell lines confirmed cross resistance of the anti-tumor effects of the two cytidine analogues. Second doses of 5-aza-CR, with the timing og administration based upon the differing patterns of recovery of [³H]TdR incorporation between normal tissues and tumor cells, led to a prolongation of survival in mice bearing the sensitive L1210 ascites tumor.     

Mu-opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPgammaS binding assays

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K(d) 3. 8+/-0.18 nM) to membranes of rat brain affording a B(max) of 87. 1+/-4.83 fmol/mg. Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for delta- ([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa- (5alpha,7alpha, 8beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPgammaS binding. The EC(50) value of morphine increased about 500-fold in the presence of 10 microM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization.     

ACTH and the stress-induced changes of lysine incorporation into brain and liver proteins.

When mice were subjected to footshock treatment and subsequently injected with [3H] lysine, the cerebral uptake of [3H] lysine, its incorporation into brain protein and the relative radioactivity (RR = protein radioactivity divided by amino acid radioactivity) were all increased. In the liver, footshocked mice showed decreased free lysine radioactivity, and increased protein radioactivity and relative radioactivity compared to quiet mice. The possibility that ACTH mediated these effects was investigated. The injection of saline had no effect in the brain but partially mimicked the footshock responses in the liver. Injections of ACTH 1--24 mimicked the effects of footshock in the brain, and further augmented the saline-induced effect on the RR in the liver. ACTH 4--10 increased the RR of brain protein, but produced no significant change in brain free lysine radioactivity or in any measure in the liver. Pretreatment of mice with the synthetic glucocorticoid, dexamethasone, did not enhance these effects and diminished the effect of ACTH 4--10 in the brain. ACTH treatment did not alter the profiles of brain polyribosomes. Lysine vasopressin, which is also released during stress, did not alter the incorporation of [3H] lysine into brain or liver protein, except at high doses when it decreased plasma radioactivity. These results suggest that secretion of ACTH at least partially mediates the stress-induced changes of [3H] lysine incorporation into brain and liver proteins, but that it is probably not the only factor involved.     

Characterization of raloxifene glucuronidation in vitro: contribution of intestinal metabolism to presystemic clearance.

Raloxifene, a selective estrogen receptor modulator used for the treatment of osteoporosis, undergoes extensive conjugation to the 6-beta- and 4'-beta-glucuronides in vivo. This paper investigated raloxifene glucuronidation by human liver and intestinal microsomes and identified the responsible UDP-glucuronosyltransferases (UGTs). UGT1A1 and 1A8 were found to catalyze the formation of both the 6-beta- and 4'-beta-glucuronides, whereas UGT1A10 formed only the 4'-beta-glucuronide. Expressed UGT1A8 catalyzed 6-beta-glucuronidation with an apparent K(m) of 7.9 microM and a V(max) of 0.61 nmol/min/mg of protein and 4'-beta-glucuronidation with an apparent K(m) of 59 microM and a V(max) of 2.0 nmol/min/mg. Kinetic parameters for raloxifene glucuronidation by expressed UGT1A1 could not be determined due to limited substrate solubility. Based on rates of raloxifene glucuronidation and known extrahepatic expression, UGT1A8 and 1A10 appear to be primary contributors to raloxifene glucuronidation in human jejunum microsomes. For human liver microsomes, the variability of 6-beta- and 4'-beta-glucuronide formation was 3- and 4-fold, respectively. Correlation analyses revealed that UGT1A1 was responsible for 6-beta- but not 4'-beta-glucuronidation in liver. Treatment of expressed UGTs with alamethicin resulted in minor increases in enzyme activity, whereas in human intestinal microsomes, maximal increases of 8-fold for the 6-glucuronide and 9-fold for the 4'-glucuronide were observed. Intrinsic clearance values in intestinal microsomes were 17 microl/min/mg for the 6-glucuronide and 95 microl/min/mg for the 4'-isomer. The corresponding values for liver microsomes were significantly lower, indicating that intestinal glucuronidation may be a significant contributor to the presystemic clearance of raloxifene in vivo.     

Influence of anticoagulants on growth and vitamin K production of intestinal bacteria.

The influence of anticoagulants on growth and vitamin K production of intestinal bacteria was studied. Bacteroides vulgatus and Escherichia coli were grown in vitro in the presence of increasing amounts of warfarin, phenprocoumon and acenocoumarol. It was found that growth of B. vulgatus was inhibited under anaerobic conditions whereas growth of E. coli under aerobic conditions was not inhibited. A specific inhibition of vitamin K biosynthesis was not observed in either case. It is concluded that therapeutic doses of anticoagulants are unlikely to affect growth or vitamin K production of intestinal bacteria.     

Synthesis of acyl[35S]sulfonamides: Coupling of high specific activity [35S]methane sulfonamide with acids and acid chlorides

Direct coupling of high specific activity [³⁵S]methanesulfonamide, generated from [³⁵S]methanesulfonyl chloride and ammonia, with acids and acid chlorides afforded the corresponding [³⁵S]acyl sulfonamides in excellent yields. Examples of high specific activity [³⁵S]acyl sulfonamides were prepared containing functionality that can be further elaborated through carbon–carbon or carbon–nitrogen bond forming reactions.     

Studies on in vitro metabolism of acrylamide and related compounds

The in vitro biotransformations of acrylamide and ten related compounds in the hepatic enzyme system of the mouse were studied in order to learn more about their toxic actions in vivo. Of nine analogues, which could be analyzed quantitatively by gas chromatography, seven compounds — N-tert-butylacrylamide, diacetone acrylamide, N,N-dimethylacrylamide, N-isobutoxymethylacrylamide, N-isopropylacrylamide, methacrylamide, and N-methylacrylamide — were metabolized in microsomal enzymes with a NADPH generating system. One or two metabolites from each of the seven compounds, except for N-isobutoxymethylacrylamide, were detected by gas chromatography. The metabolite of N-isopropylacrylamide was identified as acrylamide by gas chromatography-mass spectrometry. The metabolite of N,N-dimethylacrylamide showed a RT value identical with and a mass spectrum similar to N-methylacrylamide. No metabolites from the other four compounds have yet been identified. Acrylamide and crotonamide did not seem to be metabolized in the same system. Phenobarbital pretreatment of mice enhanced the metabolic reactions of the seven compounds, but did not elevate those of acrylamide and crotonamide. The Km value of N-isopropylacrylamide was 0.35 mM, which was the smallest of all the test analogues. All of the eleven analogues studied were found to be metabolized by hepatic glutathione S-transferases as well. This reaction was also elevated by the phenobarbital treatment of mice. The relationships between the in vitro metabolisms and the in vivo toxicities of acrylamide analogues are discussed.     

Alteration of amino acid incorporation into proteins of the nervous system in vitro after administration of acrylamide to rats

Incorporation in vitro of either l-[U-¹⁴C]lysine or l-[³⁵S] methionine into proteins in isolated nervous tissues from rats treated with acrylamide has been determined. Incorporation of the amino acids was different in different tissues. Both potassium cyanide and 2,4-dinitrophenol inhibited the incorporation, suggesting that the incorporation is dependent on aerobic energy metabolism. In the brain cortex and liver the incorporation of amino acids was not affected by acrylamide, while in the spinal cord it started to increase after the rats became paralytic, and it reached the maximum value after acrylamide was withdrawn. In contrast, in sciatic nerve, lysine incorporation was suppressed at the early stage but increased later as in the spinal cord. Methionine incorporation into sciatic nerve was not decreased. Equilibration of lysine between the suspending medium and the tissue both in control and treated rats was established within at least 10 min after the incubation was begun. Autoradiographically a large number of silver grains due to [¹⁴C]lysine was visible in the anterior horn cells of the spinal cord and in the Schwann cells of the sciatic nerve both in normal and treated rats. A possible mechanism for the altered protein metabolism is discussed.     

Effect of basic cholane derivatives on intestinal cholic acid metabolism: in vitro and in vivo activity.

A representative series of hydroxy-5 beta-cholanyl-24-amines were tested both in vitro and in vivo with respect to their activity against the intestinal bacteria responsible for bile acid metabolism. For the in vitro studies, radiolabeled [14C]cholic acid was incubated with human stools both in aerobic and anaerobic conditions in the presence of the title compounds at a dose of 10 micrograms/mL, and the biotransformation of cholic acid into radiolabeled deoxycholic acid and other metabolites was followed by TLC-radiochromatography. Of the compounds studied, 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-N-methylamine showed the highest activity. This compound was used for the in vivo studies and was shown to inhibit the formation of endogenous secondary bile acids when chronically administered to rats at a dose of 60 micrograms/day for 15 days. The treated rats showed an increased ratio of taurocholic acid (primary bile acid) to taurodeoxycholic acid (secondary bile acid) in bile, a fact further suggesting a potent antibacterial activity of the compound toward bacteria responsible for bile acid metabolism.     

Effect of thiocarbonyl compounds on alpha-naphthylisothiocyanate-induced hepatotoxicity and the urinary excretion of [35S]alpha-naphthylisothiocyanate in the rat

The effect of disulfiram (DSF), sodium diethyldithiocarbamate (DDTC), methyl diethyldithiocarbamate (Me-DDTC), and ethionamide on the hepatotoxic response of alpha-naphthylisothiocyanate (ANIT) was studied in the rat. The hyperbilirubinemic response of ANIT was significantly inhibited by ip or po DSF pretreatment. A more marked inhibition of toxicity occurred when DSF was given via ip injection. DDTC, Me-DDTC, and ethionamide significantly inhibited ANIT-induced hyperbilirubinemia. Me-DDTC is approximately three times more potent than DDTC as an inhibitor of toxicity. Approximately 16% of a dose of [35S]ANIT was excreted in the urine as inorganic sulfate 48 hr after dosing. Me-DDTC administered simultaneously with [35S]ANIT significantly reduced urinary [35S]sulfate excretion in the first 24 hr. Ethionamide reduced urinary [35S]sulfate excretion. Pretreatment with phenobarbital which stimulates toxicity in vivo increased urinary [35S]sulfate excretion 300% in the first 12 hr. Thus, this study shows that agents which sensitize or protect rats from the toxic effects of ANIT, correspondingly stimulate or inhibit the oxidative desulfuration of [35S]ANIT in vivo.