Dissipation by tetraethylammonium of verapamil-stimulated p-aminohippurate accumulation in rat kidney cortical slices

The stimulatory effect of verapamil on p-aminohippurate (PAH) accumulation was studied in rat kidney cortical slices. PAH accumulation was stimulated by the presence of 0.3 mM verapamil in the incubation medium, but this stimulatory action of verapamil was dissipated by adding tetraethylammonium (TEA) to the medium. TEA accumulation by the kidney cortical slices was inhibited by verapamil in a concentration-dependent fashion. In addition, kinetic studies revealed that verapamil resulted in an increase in the Michaelis constant (K_m) of TEA transport, with the V_max remaining constant, suggesting that verapamil competitively inhibited TEA transport. These results suggest that the transport system for verapamil is the same as that for TEA and that verapamil taken up within the cells may stimulate PAH transport.     

Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-1,3-butadiene and its conjugates

Hexachloro-1,3-butadiene (HCBD), its glutathione conjugate (HCBD-GSH), cysteine conjugate (HCBD-CYS), and mercapturic acid derivative (HCBD-NAC) all produce acute necrosis of the pars recta of the proximal renal tubule in the rat. Previous studies have shown that radiolabel from administered HCBD appears to concentrate in the pars recta region. Renal uptake of radioactivity from HCBD-NAC was studied in rats by giving a single ip injection of the chemical and measuring its concentration in plasma and renal cortex 4 hr later. Cortex/plasma ratios (C/P) of HCBD-NAC were 4.35 +/- 0.21 (8 animals) at a dose of 64 mumol/kg and 10.4 +/- 0.55 (5) at a dose of 16 mumol/kg. These ratios were greater than that of inulin [C/P inulin = 1.5 +/- 0.2 (4)]. Thus cortical HCBD-NAC content was significantly greater than can be accounted for by glomerular filtration alone. Prior administration of probenecid (500 mumol/kg), a competitive inhibitor of organic acid transport, to animals receiving 16 or 64 mumol/kg of HCBD-NAC reduced the C/P to 1.03 +/- 0.09 (5) and 0.81 +/- 0.05 (8), respectively. Administration of probenecid in increasing doses (100, 200, 300, and 400 mumol/kg) to animals receiving 64 mumol/kg HCBD-NAC resulted in decreases of the C/P (2.59, 2.29, 1.35, and 0.84, respectively), suggesting a competitive inhibition of cortical HCBD-NAC uptake. The extent of covalently bound radioactivity from 64 mumol/kg HCBD-NAC was significantly greater in the renal cortex (1.11 +/- 0.2 nmol eq/mg protein) than in the liver (0.19 +/- 0.01 nmol eq/mg protein). Prior administration of probenecid (500 mumol/kg) reduced the renal cortical concentration of HCBD-NAC to 0.25 +/- 0.02 nmol eq/mg protein. Increasing doses of probenecid resulted in a progressive decrease in renal cortical covalent binding. When treatment with probenecid led to renal cortical concentrations of less than 120 nmol eq HCBD-NAC/g and an amount of covalently bound material less than 0.4 nmol eq/mg protein the animals were completely protected against the nephrotoxicity, as assessed by plasma urea and histopathological examination 24 hr after dosing. Prior administration of probenecid (500 mumol/kg) also protected rats against the nephrotoxicity produced by HCBD (192 mumol/kg), HCBD-GSH (47 mumol/kg), and HCBD-CYS (36 mumol/kg). It is suggested that the renal cortical accumulation and selective proximal tubular toxicity of HCBD and its conjugates is related to a carrier-mediated transport system.     

Carrier-mediated transport of amino-cephalosporins by brush border membrane vesicles isolated from rat kidney cortex

The uptake of cephalosporin antibiotics by brush border membrane vesicles isolated from rat renal cortex has been studied by a rapid filtration technique, demonstrating a carrier-mediated transport system for amino-cephalosporins such as cephalexin and cephradine. The antibiotics were taken up into an osmotically reactive intravesicular space. The uptake of cephalexin was saturable (apparent Km2.2 mM), was inhibited by structural analogues and sulfhydryl reagents, and was stimulated by the countertransport effect, although the Na+ gradient did not affect the uptake. This transport system was essentially different from the transport system for p-aminohippurate in brush border membranes. The uptake properties for cephradine in brush border membrane vesicles appeared to be similar to those for cephalexin. The present results suggest the existence of a carrier-mediated transport system for amino-cephalosporins in brush border membranes. This system may be a part of the mechanism of tubular reabsorption of these antibiotics.     

The effects of phenformin on the transport and metabolism of sugars by the rat small intestine

The effects of 0.25–10 mM phenformin on sugar transport and metabolism have been studied in a preparation for the combined perfusion of the vascular bed and the lumen. At all concentrations the effects of vascular phenformin were more pronounced than those of luminal phenformin. Phenformin inhibited galactose transport across the intestine, the pattern of inhibition depending on whether the phenformin was added to the luminal or vascular compartments. The active accumulation of galactose in the mucosal epithelial cells was also abolished. There was a linear relationship between the percentage reduction in mucosal ATP levels and vascular phenformin concentration. Phenformin reduced the rate of glucose uptake from the lumen, and the proportion of this glucose which reached the vascular effluent. Most of the glucose which did not reach the vascular side could be accounted for by the formation of lactic acid. Vascular phenformin increased glucose uptake from the vascular medium by ca 88%, 97% of which could be accounted for by lactate formation. Phenformin was sequestered by the mucosa when added to the vascular, but not the luminal, perfusates. There was very little translocation of intact phenformin across the gut in either the mucosal or serosal directions. It is suggested that the effects of phenformin on the gut mainly derive from an inhibition of mitochondrial oxidative phosphorylation, with a small contribution from a direct effect on the brush border, more pronounced at high phenformin concentrations. The results are consistent with the idea that phenformin delays sugar absorption in man, and that the intestine may be a significant source of lactate production in lactic acidosis     

Effect of substituents on in vitro metabolism and covalent binding of substituted bromobenzenes.

The relative hepatotoxicity of o-bromobenzonitrile, bromobenzene, o-bromotoluene, and o-bromobenzotrifluoride decreases in the order mentioned. In this study, the nature of the substrate-enzyme interaction was investigated by determining spectral binding constants and by following the disappearance of several substituted bromobenzenes in vitro in the presence of the hepatic monooxygenase system. The disappearance rates generally parallel substrate electronic character; all the compounds display spectral binding constants of similar magnitude, and differences are not obviously attributable either to substituent solubility or electronic properties. In addition, the extent to which metabolites of these materials become covalently bound to microsomal proteins following incubation in vitro was determined. Despite an apparently low rate of metabolism, the very toxic o-bromobenzonitrile was covalently bound to a greater extent than bromobenzene. Surprisingly, the less toxic o-bromotoluene was the most efficiently covalently bound substrate studied. Whereas the extent of covalent binding of bromobenzene is increased ninefold by pretreating the animals with phenobarbital, only a 2.4-fold enhancement of o-bromobenzonitrile binding was observed. Glutathione reduced the covalent binding observed for all substrates in concentrations as low as 0.01 mm (except o-bromobenzotrifluoride, which required 0.1 mm). The in vivo metabolism of o-bromotoluene was also studied, and it was determined that the major urinary metabolites are phenols and mercapturic acids, products of the arene oxide metabolic pathway. There does not emerge a simple correlation between macromolecular covalent binding and toxicity among these compounds.     

Isolation of naphthazarin from walnut "onigurumi" and its inhibitory action on oxidative phosphorylation in mitochondria

Investigation of lethal substances other than juglone in the walnut ‘Onigurumi’, Juglans mandshurica maxim var. Sieboldiana Makino, led to the isolation and identification for the first time, of 5,8-dihydroxy-1,4-naphthoquinone (naphthazarin) in plants. Toxicity experiments with both these fish toxins on albino orange-red killifish ‘Himedaka’, Oryzias latipes, gave median tolerance limits (TL_m) of 0.245 and 0.435 ppm, respectively. A study on the inhibitory effect of these toxins on oxidative phosphorylation showed half maximal inhibitin values ranging between 10^-4 and 10^-5 M for naphthazarin and between 10^-5 and 10^-6 M for juglone. Naphthazarin had complicated dualistic effect acting both as an uncoupler and energy transfer inhibitor, while juglone was only an energy transfer inhibitor.     

Preparation and characterization of subcellular fractions suitable for studies of drug metabolism from the trunk kidney of the Northern pike (Esox lucius) and assay of certain enzymes of xenobiotic metabolism in these subfractions

The present study was designed to prepare and characterize subcellular fractions from the trunk kidney of the Northern pike (Esox lucius), with special emphasis on the preparation of a microsomal fraction suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. Finally, the subcellular distributions of several drug-metabolizing enzymes (NADPH-cytochrome c reductase, NADH-ferricyanide reductase, glutathione transferase, epoxide hydrolase) were determined. With the exception of NADPH-cytochrome c reductase, the subcellular distributions obtained here for drug metabolizing and marker enzymes closely resembled those reported for rat liver. NADPH-cytochrome c reductase was apparently partially solubilized here from microsomal vesicles by an endogenous protease, which reduced its usefulness as a marker enzyme and raises questions concerning the measurement of activities catalyzed by the cytochrome P-450 system in these subfractions. In other respects the microsomes and supernatant fraction prepared here from the trunk kidney of the pike seem to be as well suited for investigations of drug metabolism as are the corresponding fractions from rat and pike liver.     

Effect of deuteration of imipramine on its pharmacokinetic properties in the rat

Imipramine was specifically deuterated in either both aromatic rings or in the N-methyl group, or in both positions, and the pharmacokinetic properties of the products were determined in the rat and compared with those of the non-deuterated analogue. Deuteration of imipramine resulted in a small but significant isotope effect on N-demthylation while aromatic hydroxylation was unaffected. This isotope effect led to a slower rate of systemic clearance, a longer half-life and, when orally administered, enhanced bioavailability. Urinary excretion of didesmethylimipramine-d₄, following oral administration of imipramine-d₇, was significantly lower than the excretion of didesmethylimipramine following administration of unalabelled imipramine, indicating inhibited demethylation. Similarly, the urinary excretion of desmethylimipramine-d₄, didesmethylimipramine-d₄ and 2-hydroxydes-methylimipramine-d₄ were lower than for the corresponding unlabelled or d₇-analogues, indicating the stability of the N-CD₃ group. Deuteration had no effect on the pharmacological properties of imipramine as determined in this study.     

Interactions of ethanol with ionophore A23187 in human platelets and erythrocytes and in rat brain slices

The aggregation of gel-filtered human platelets induced by A23187 is very sensitive to inhibition by ethanol. Similarly when platelets preloaded with [3H]5-hydroxytryptamine ([3H]5HT) are studied in a superfusion system under conditions where aggregation is likely (high platelet density, presence of Ca2+) the rate of release of [3H]5HT induced by A23187 is reduced by the presence of ethanol. However when platelet aggregation is less likely (low platelet density, absence of Ca2+) ethanol does not reduce the rate of [3H]5HT efflux induced by A23187 in superfused platelets. In addition, in contrast to the effects of ethanol on platelet aggregation, the transformation of human red cells to echinocytes induced by A23187 is accelerated by the presence of ethanol. Similarly the increased efflux of 3H from superfused rat striatal slices preloaded with [3H]dopamine which is produced by A23187 is potentiated by ethanol. It is concluded that the inhibitory effect of ethanol on the action of A23187 may be confined to platelet aggregation. This may be because the mechanisms of action of either A23187 or ethanol on platelet aggregation differ from those on other cell functions.     

Effect of various metabolic inhibitors on biphenyl metabolism in isolated rat hepatocytes.

The effect of three inhibitors of mitochondrial function (menadione, rotenone and 2,4-dinitrophenol) on drug metabolism in isolated rat hepatocytes has been studied. Menadione (at 1.25 × 10^?4 M) caused almost complete inhibition of biphenyl Phase I metabolism whereas rotenone (2 × 10^?5 M) inhibited the same reaction only by 25 per cent although the subsequent conjugation of the Phase I metabolite was markedly depressed. Qualitatively similar findings were observed with hepatocytes isolated from phenobarbital-pretreated rats, and with liver microsomes isolated from control rats. 2,4-Dinitrophenol (2 × 10^?4 M) caused a marked enhancement of biphenyl Phase I metabolism but a marked inhibition of subsequent conjugation. This enhancement of Phase I metabolism was not observed in control cells with other substrates (benzo[a]pyrene, 7-ethoxycoumarin), nor in biphenyl metabolism in “induced” cells or in liver microsomes isolated from control rats. It is tentatively suggested that products of 2,4-dinitrophenol metabolism may “activate” biphenyl metabolism in intact liver cells. Furthermore, it is suggested for all three inhibitors that direct effects on the drug metabolizing enzyme systems (Phase I and Phase II) are as important as their effects on mitochondrial function in explaining their inhibition of drug metabolism. It appears that Phase II metabolism of xenobiotics is more susceptible to inhibition by metabolic inhibitors than is Phase I metabolism, probably due to depletion of the cellular ATP levels.     

Phenolphthalein- and harmaline-induced disturbances in the transport functions of isolated brush border and basolateral membrane vesicles from rat jejunum and kidney cortex

Phenolphthalein and harmaline were examined with respect to their effects on the transport functions of purified brush border and basolateral membrane vesicles from rat jejunum and kidney cortex. Phenolphthalein (0.5 mM) inhibited Na⁺-coupled d-glucose uptake by intestinal brush border membrane vesicles without affecting Na⁺-coupled l-alanine transport, Na⁺ transport or Na⁺-independent d-glucose transport. In renal brush border membrane vesicles, the same concentration of this drug did not even affect Na⁺-coupled d-glucose uptake. At a concentration of 1 mM or higher, however, Phenolphthalein rendered both intestinal and renal vesicles leaky to these solutes. In intestinal and renal basolateral membrane vesicles, phenolphthalein at a concentration of 0.5 mM noticeably inhibited (Na⁺-K⁺)-ATPase activity, but showed no effect on phloretin-sensitive Na⁺-independent d-glucose uptake. At 1 mM this drug also inhibited ouabain-insensitive ATPase activity. Harmaline, at concentrations greater than 2 mM, inhibited not only Na⁺-coupled d-glucose and l-alanine uptake by both intestinal and renal brush border membrane vesicles, but also Na⁺ translocation. The drug, however, affected neither Na⁺-independent d-glucose uptake nor the general permeability of these membranes. Harmaline also inhibited (Na⁺-K⁺)-ATPase activity of intestinal and renal basolateral membrane vesicles without affecting ouabain-insensitive ATPase. It did not influence, however, phloretin-sensitive Na⁺-independent d-glucose uptake by these vesicles. These observations suggest that harmaline acts as an inhibitor of Na⁺ and Na⁺-dependent transport mechanisms in intestinal as well as renal brush border membranes. Phenolphthalein at the lower concentration selectively inhibited certain transport processes in these membranes as well as in basolateral membranes, whereas at the higher concentration it caused widespread structural disturbances, possibly through its chaotropic action on membranes.     

Toxicity and metabolism of malathion and its impurities in isolated rat hepatocytes: Role of glutathione

The role of glutathione (GSH) in the detoxification of malathion and in the potentiating action of impurities on the toxicity of malathion was investigated in freshly isolated rat hepatocytes. Malathion and four of its major impurities at concentrations of 0.01 to 1 mm caused a marked depletion of the hepatocellular GSH content within 60 min. The potency of the compounds to deplete the cellular GSH level declined in the following order: isomalathion = O,S,S-trimethyl phosphorodithioate > malathion=O,O,S-trimethyl phosphorodithioate >O,O,S-trimethyl phosphorothioate. Preexposure of hepatocytes to 0.03 mm of isomalathion or O,S,S-trimethyl phosphorodithioate enhanced the GSH-dependent detoxification of malathion up to threefold. When carboxylesterase activity was inhibited, the resulting depletion of the GSH level by more than 70% greatly potentiated the toxicity of malathion as measured by the inhibition of cholinesterase activity. These experiments indicate that the simultaneous blockage of the carboxylesterase- and GSH-dependent routes of malathion detoxification is a possible mechanism for the potentiating action of impurities on the malathion toxicity in mammals.     

Effects of hexafluoroacetone on testicular morphology and lipid metabolism in the rat

Rats dosed dermally with 39 or 130 mg/kg/day hexafluoroacetone sesquihydrate (HFA) for 14 days developed moderate or severe testicular atrophy, respectively; rats dosed with 13 mg/kg/day HFA for 14 days did not. Histologic evaluation of the testes revealed that spermatids, followed by spermatocytes, were the germ cells most affected by HFA; spermatogonia and Sertoli cells appeared to be less vulnerable. Lipogenesis from [³H]acetate and [¹⁴C]glucose was investigated in vitro in testes from HFA-treated and pair-fed control rats. Triacylglycerol and phospholipid synthesis was increased whereas sterol synthesis was decreased in testes from HFA-treated rats. Vitamin A and zinc were measured in the testes of control and HFA-treated rats; no differences in the levels of these nutrients were observed between the two groups. The data support the hypothesis that altered lipid metabolism, in particular sterol metabolism, is associated with the development of HFA-induced testicular atrophy.     

Studies of the mode of action of antitumour triazenes and triazines—V. The correlation of the in vitro cytotoxicity and in vivo antitumour activity of hexamethylmelamine analogues with their metabolism

Experiments were conducted to ascertain whether the antitumour activity of hexamethylmelamine analogues correlated with their in vitro cytotoxicity and metabolism. Two analogues, namely pentamethylmelamine (PMM) and 2,2,4,4-tetramethylmelamine (TMM), and hexamethylmelamine (HMM) itself were shown to be active towards the murine ADJ/PC6A (PC6) plasmacytoma; another three, 2-chloro-4,6-bis(dimethylamino)-1,3,5-triazine (CBDT), 2,4-bis-(dimethylamino)-6-hydrazino-1, 3,5-triazine (HBDT) and 2,4,6-trimethylmelamine (TriMM) were inactive against the same tumour. The cytotoxicity of these compounds was examined against a PC6 tumour cell line in vitro. In the absence of liver microsomal activation only CBDT proved to be significantly cytotoxic at a concentration of 5 mM. In the presence of murine liver microsomes the three active antitumour agents were all cytotoxic at this concentration whereas HBDT and TriMM remained non-toxic. The degree of cytotoxicity correlated with the extent of metabolism for these analogues. The products of biotransformation of these compounds were stable precursors of formaldehyde (presumably N-hydroxymethyl intermediates) (FP) rather than formaldehyde itself. After injection of these 6 compounds to Balb/c mice the levels of FP generated in the plasma were markedly greater for the three active antitumour agents than for the inactive analogs. No free formaldehyde was detected in the plasma after administration of any of the compounds.These results suggest that for these compounds in vitro cytotoxicity correlates with in vitro biotransformation and their antitumour activity correlates with plasma levels of FP generated by metabolism in vivo.     

The metabolism and disposition of hexachloro-1:3-butadiene in the rat and its relevance to nephrotoxicity

Following po administration of a nephrotoxic dose (200 mg/kg) of hexachloro-1:3-butadiene (HCBD) to male rats, the principal route of excretion was biliary, 17-20% of the dose being eliminated on each of the first 2 days. Fecal excretion over this period was less than 5% of the dose per day, suggesting enterohepatic recirculation of biliary metabolites. Urinary excretion was small, not exceeding 3.5% of the dose during any 24-hr period. The major biliary metabolite was a direct conjugate between glutathione and HCBD itself. The cysteinylglycine conjugate of HCBD has also been found in bile. Evidence was obtained to show that biliary metabolites of HCBD are reabsorbed and excreted via the kidneys. The glutathione conjugate, its mercapturic acid derivative, and bile containing HCBD metabolites were all nephrotoxic when dosed orally to rats. In common with HCBD, these metabolites caused localized damage to the kidney with minimal effects in the liver. Rats fitted with a biliary cannula were completely protected from kidney damage when dosed with HCBD, demonstrating that hepatic metabolites were solely responsible for the nephrotoxicity of this compound. It is proposed that the hepatic glutathione conjugate of HCBD was degraded to its equivalent cysteine conjugate which was cleaved by the renal cytosolic enzyme beta-lyase to give a toxic thiol which caused localized kidney damage. A urinary sulphenic acid metabolite of HCBD has been identified which is consistent with this hypothesis. The mode of activation of HCBD conjugates in the kidney is believed to be analogous to that proposed for S-(1,2-dichlorovinyl)-L-cysteine.     

Non-ionic surfactants and the membrane transport of barbiturates in goldfish

The effect of a series of polyoxyethylene non-ionic surfactants on the membrane transport of barbiturates in goldfish has been assessed using the overturn time technique. The surfactants, ranging in polyoxyethylene chain length from 2 to 60 and alkyl chain length from 4 to 18, were used at concentrations ranging from 0.001 to 0.1% w/v. Presence of the surfactants in the bathing medium caused a concentration-dependent alteration in the absorption of secobarbitone and thiopentone. The greatest enhancement of absorption for each barbiturate occurred with surfactants with 10 to 20 ethylene oxide moieties, alkyi chain lengths of C₁₂-C₁₆ and molecular areas of between 1.00 and 1.60 nm². The results are comparable with data obtained with the same surfactants using other epithelial membranes.