Effects of phenobarbital and 3-methylcholanthrene on the in vivo distribution,metabolism and covalent binding of 4-ipomeanol in the rat; implications for target organ toxicity

The effects of phenobarbital (PB) and 3-methylcholanthrene (MC) on the distribution, metabolism and covalent binding of 4-ipomeanol were examined in the rat. An analysis of tissue extracts by high-pressure liquid chromatography (HPLC) showed that both treatments markedly decreased the concentrations of unmetabolized 4-ipomeanol at all times examined. PB treatment increased the urinary excretion of nonbound 4-ipomeanol metabolites, while MC treatment did not alter their excretion. Analysis of urine by HPLC indicated that the increased concentration of urinary metabolites found in the phenobarbital-treated rats was attributable primarily to an increased excretion of ipomeanol-4-glucuronide. These data indicate that the decreased pulmonary covalent binding and lethality of 4-ipomeanol in the rat after MC and PB were caused by alterations in the tissue distribution of the parent compound. Pulmonary concentrations of unmetabolized 4-ipomeanol were decreased by MC through an increased metabolism of 4-ipomeanol in the liver, primarily to toxic products that bind covalently in that tissue and lead to hepatotoxicity. PB produced a similar decrease in unmetabolized 4-ipomeanol concentrations in lung but by an enhanced in vivo metabolism and clearance of 4-ipomeanol, primarily through a “nontoxic” pathway, glucuronidation, and did not lead to hepatotoxicity.     

Further studies of stereospecificity at carbon 6 for membrane transport of tetrahydrofolates: Diastereoisomers of 5-methyltetrahydrofolates as competitive inhibitors of transport of methotrexate in L1210 cells

The unnatural d diastereoisomer at carbon 6 of 5-methyltetrahydrofolate was only slightly less effective than the natural l diastereoisomer as a competitive inhibitor of the carrier-mediated membrane transport of [³H]methotrexate into L1210 murine leukemia cells. The apparent K_i for a mixture containing equal amounts of both natural and unnatural diastereoisomers was not significantly different from that found for the unnatural form. These results show that the reduced folate carrier system in these cells has a strong affinity for the unnatural stereoisomer, a finding in contrast to that obtained with the corresponding diastereoisomer of 5-formyltetrahydrofolate.     

Cytotoxicity of isoproterenol to cultured heart cells: effects of antioxidants on modifying membrane damage

Primary cultures of rat myocytes were used to study the cardiac damage induced by toxic doses of L-isoproterenol (ISO). Cultures were exposed to varying concentrations of ISO (2.4 X 10(-5), 1 X 10(-4), and 5 X 10(-4) M) for 0.5, 1.5, 4, and 12 hr. Mitochondrial membrane fragility, myocyte potassium content (as an index of sarcolemmal damage), and cellular glutathione content were used to evaluate cellular injury. A significant increase in mitochondrial fragility was observed 0.5 hr after treatment with 5 X 10(-4) M ISO. Lower doses caused an increase in mitochondrial fragility 1.5 hr after exposure. Longer durations of ISO exposure (4 and 12 hr) were necessary to decrease cellular potassium content. Glutathione levels were minimally affected by ISO. L-Ascorbic acid (5 X 10(-3) M) or sodium bisulfite (9.6 X 10(-4) M) were added to the cultures to determine if antioxidants prevent the toxicity caused by ISO. The presence of L-ascorbic acid or sodium bisulfite in ISO-treated myocyte cultures prevented the toxic changes in mitochondrial fragility and myocyte potassium content. The data indicate that antioxidants may be useful in reducing injury induced by toxic doses of ISO. Furthermore, mitochondrial injury may be involved significantly with the development of ISO-induced cardiotoxicity.     

The competitive inhibition of glucose transport in human erythrocytes by compounds of different structures

By kinetic analysis we found that the transport protein for glucose in human erythrocyte membranes has different binding sites for competitive inhibitors. They all change the transport protein with the effect that it loses its affinity to glucose. Some of the competitive inhibitors alter the conformation of the transport protein, so that other ones cannot be boand. There are inhibitors, however, which do not affect the affinity of other competitive inhibitors. A schematic model of our assumption about the mechanism of the competitive inhibition of glucose transport is presented.     

Protective role of endogenous pulmonary glutathione and other sulfhydryl compounds against lung damage by alkylating agents: Investigations with 4-ipomeanol in the rat

Because endogenous glutathione is known to participate in the detoxification of highly reactive, hepatotoxic drug metabolites, we studied the role of this substance in the pulmonary toxicity of 4-ipomeanol [1-(3-furyl)-4-hydroxypentanone] in rats. 4-Ipomeanol was an appropriate model for these studies since previous investigations have indicated that an alkylating metabolite, formed in situ, is responsible for selective lung damage by 4-ipomeanol. Toxic doses of 4-ipomeanol preferentially depleted rat lung glutathione. Pretreatment of animals with piperonyl butoxide, an inhibitor of the metabolic activation of 4-ipomeanol, prevented both the depletion of lung glutathione and the pulmonary toxicity of 4-ipomeanol. Prior depletion of lung glutathione by diethylmaleate increased both the pulmonary covalent binding and the toxicity of 4-ipomeanol, whereas administration of cysteine and cysteamine decreased both the covalent binding and the toxicity. These in vivo studies, in conjunction with previous in vitro studies which showed inhibitory effects of sulfhydryl compounds on the covalent binding of 4-ipomeanol, are consistent with the view that pulmonary glutathione plays a protective role against pulmonary alkylation and lung toxicity by 4-ipomeanol, probably by reacting with the toxic metabolite(s) to form nontoxic conjugate(s). Pulmonary glutathione may similarly provide protection against other electrophilic drugs or metabolites that can damage the lungs.     

Stimulation by adriamycin of rat heart and liver microsomal NADPH-dependent lipid peroxidation

Rat liver and heart microsomes catalyze the transfer of single electrons from NADPH to adriamycin forming semiquinone radicals which, in turn, activate molecular oxygen. This process stimulated lipid peroxidation 5- to 7-fold as measured by malonaldehyde formation. Adriamycinaugmented lipid peroxidation was linear with time to 60 min, optimal at 1.0 mg of microsomal protein/ml and pH 7.5, and was proportional to the adriamycin concentration up to 100 μM. An NADPH-generating system was superior to NADPH, and an oxygen atmosphere tripled the rate of peroxidation as compared to air. Nitrogen abolished adriamycin-stimulated peroxidation. Superoxide dismutase, reduced glutathione, α-tocopherol, EDTA, dioxopiperazinylpropane (ICRF-187), and dimethylurea were effective inhibitors of lipid peroxidation. This suggests that Superoxide anion and possibly hydroxyl radical may be formed by the oxidation of the adriamycin semiquinone radical and thus stimulate the peroxidation of microsomal unsaturated fatty acids. Although adriamycin failed to stimulate lipid peroxidation in heart microsomes from control animals, peroxidation was dramatically increased when adriamycin was added to cardiac microsomes from α-tocopherol-deficient rats. Lipid peroxidation in α-tocopheroldeficient liver microsomes was four times greater than in control microsomes with the NADPH-generating system, and adriamycin did not further increase that high rate of peroxidation; however, when NADPH was used as the source of electrons in place of the NADPH-generating system, adriamycin stimulated peroxidation more than 2-fold. These results suggest that microsomal lipid peroxidation may play a role in the cytotoxicity and cardiotoxicity of adriamycin.     

Reduction in eumelanin by the activation of glutathione reductase and gamma-glutamyl transpeptidase after exposure to a depigmenting chemical

Topical application of 4-tertiary butyl catechol (TBC) causes vitiligo in the skin of man and animals, and previous electron microscopic studies showed pheomelanin formation in the affected areas. In the present study, we investigated changes of enzyme activities, eumelanin content and amount of sulfur in tissue cultured human melanoma cells exposed to the depigmenting chemical. TBC enhanced glutathione reductase activity without changing the eumelanin content by 24 hr after exposure and subsequently (by 42 hr) increased gamma-glutamyl transpeptidase activity and sulfur content in the cells with a decrease in eumelanin content. It is suggested that this chemical alters the types of melanin formed by modulation of these enzyme activities.     

Relationship between cyclic AMP-dependent protein kinase activation and Ca uptake increase of sarcoplasmic reticulum fraction of hog biliary muscles relaxed by cholecystokinin-C-terminal peptides

In hog terminal bile duct cholecystokinin peptides caused an activation of cyclic AMP-dependent protein kinase (A-PK) with cyclic AMP, followed by increase in Ca uptake of sarcoplasmic reticulum fraction (SR-F). By contrast, papaverine showed no activation of A-PK-induced Ca uptake by SR-F with cyclic AMP. The Ca uptake by SR-F was dependent on ATP and Mg², but the component phosphorylated was not the phosphoenzyme intermediate in Ca²-ATPase. The effect of Ca uptake was blocked by the inclusion of a protein inhibitor of A-PK. The correlation coefficient between cyclic AMP-dependent SR-F phosphorylation and stimulated Ca uptake by the phosphorylated SR-F was 0.731 (P < 0.01). These results suggest that one of the mechanisms by which CCK-4, CCK-8, and CCK-33 peptides relax isolated Oddi's sphincters of terminal bile ducts is activation of A-PK-induced Ca uptake by sarcoplasmic reticulum fraction and possibly also by plasma membrane.     

Activation of 3,4,3′,4′-tetrachlorobiphenyl to protein-bound metabolites by rat liver microsomal cytochrome P-448-containing monooxygenase system

The specificity of two major types of cytochrome P-450 of rat liver microsomes induced by phenobarbital (PB) and 3-methylcholanthrene (MC) toward activation of three 14C-labeled tetrachlorobiphenyl (TCB) isomers to protein-bound metabolites was examined by intact microsomal and reconstituted monooxygenase systems. The monooxygenase system containing PB-inducible form(s) of cytochrome P-450 was much more active than those inducible by MC in activating two TCB isomers, i.e., 2,4,2',5'-[14C]TCB, for the binding reaction. However, the binding of 3,4,3',4'-[14C]TCB was catalyzed most actively by the system containing a MC-inducible (P-448 type) cytochrome P-450 from rat liver microsomes. Thus two forms of cytochrome P-450 had different substrate specificities for the metabolism of TCB. In another series of experiments, the site of protein molecule bound to TCB metabolites was examined by the cytochrome P-448-containing reconstituted system with 3,4,3',4'-[14C]TCB as substrate. The following lines of evidence supported the hypothesis that the active metabolites of TCB bind covalently to cysteine residues of protein molecules a) TCB-binding activity was strongly inhibited by thiol compounds such as cysteine and glutathione and b) of the various kinds of proteins tested as target for TCB metabolites, only those which contained free sulfhydryl groups on the protein molecule were radiolabeled. Moreover, we found that during the metabolism of 3,4,3',4'-TCB, the heme content of the reconstituted system was lowered, probably reflecting the alkylation of the sulfhydryl groups in the active sites of cytochrome P-448.     

Corticotropin releasing factor stimulation of protein carboxylmethylation in mouse pituitary tumor cells

A putative role for the protein carboxylmethylase (PCM) enzyme has been suggested in exocytotic secretion. The involvement of 3H-methyl incorporation into protein carboxylmethyl esters during corticotropin releasing factor (CRF)-induced ACTH secretion from AtT-20/D16-16 mouse pituitary cells was investigated. Protein carboxylmethylation and ACTH secretion both increased as a function of extracellular CRF concentration, and both processes were temporally parallel up to 60 min incubation. The less potent [Met(O)21]-CRF also stimulated increases in protein carboxylmethylation and ACTH secretion. The free acid analogue of CRF did not alter either process. A combination of the PCM inhibitors, 3-deazaadenosine and L-homocysteine thiolactone, reduced both CRF-stimulated protein carboxylmethylation and ACTH release. Dexamethasone, known to inhibit ACTH secretion and synthesis, inhibited both CRF-stimulated protein carboxylmethylation and ACTH secretion.     

Effect of phloretin on Na+-dependent D-glucose uptake by intestinal brush border membrane vesicles

In the present work we studied the effects of phloretin on Na+-dependent D-glucose uptake by brush border membrane vesicles isolated from rabbit small intestine. Phloretin had no inhibitory effect on Na+-dependent D-glucose uptake in the presence of equilibrated Na+, although it inhibited the process in the presence of a Na+ gradient. Phlorizin inhibited Na+-dependent D-glucose uptake both in the presence and in the absence of a Na+ gradient. Phloretin and phlorizin competed with each other for the inhibition of Na+-energized D-glucose uptake. These results indicate that phloretin has no direct interaction with the Na+-dependent D-glucose transporter per se, though phloretin and phlorizin may have a common step(s) in their inhibition mechanisms. Phloretin, but not phlorizin, was found to increase the equilibrium level of D-glucose uptake whether Na+ was present or not. The increase in the equilibrium level was due to phloretin-induced binding of D-glucose to the brush border membrane, but not due to an increase in the intravesicular space. It seems to occur by a mechanism different from that by which phloretin inhibits Na+-energized D-glucose uptake, because the equilibrium level of D-glucose uptake is increased by the ionized form of phloretin but Na+-energized D-glucose uptake is inhibited by the unionized form.     

Comparison of protein, RNA, and DNA binding and cell-cycle-specific growth inhibitory effects of nickel compounds in cultured cells

Crystalline NiS particles are potent inducers of morphological transformation and are actively phagocytosed by cells. Water-soluble nickel compounds are less potent, possibly because the total amount of nickel that enters cells is less, and its subcellular distribution differs in a number of ways from that following the entry of NiS in the form of an internalized particle. To further study this problem, we have examined the binding of ⁶³Ni to DNA, RNA, and protein isolated from cultured Chinese hamster ovary cells treated with either crystalline ⁶³NiS or ⁶³NiCl₂. Treatment of cultured cells with ⁶³NiS at 10 μg/ml for 3 days resulted in binding of nickel to DNA, RNA, or protein in the range of 1 μg of nickel bound per milligram macromolecule. However, similar treatment of cells with ⁶³NiCl₂ at 10 μg/ml for 1 to 5 days resulted in approximately one to several orders of magnitude less nickel bound to DNA, RNA, and protein. In the case of ⁶³NiCl₂ treatment, cellular proteins contained about 100 times more ⁶³Ni bound than the respective RNA or DNA fractions. However, the protein fraction obtained from cells treated with crystalline NiS contained about 15 times more nickel bound than the same fraction isolated from cells that were similarly treated with ⁶³NiCl₂. RNA or DNA had 300 to 2000 times more bound nickel following crystalline NiS treatment compared to cells treated similarly with NiCl₂. In contrast to the selective binding of ⁶³NiCl₂ to protein, cultured cells treated with crystalline ⁶³NiS had equivalent levels of nickel associated with RNA, DNA, and protein. Since the interaction of ⁶³Ni with these macromolecules following crystalline ⁶³NiS treatment was not due to the binding of the actual particles, the dissolution of intracellular ⁶³NiS particles probably plays an important role in governing the distribution of ⁶³Ni ions available for binding. The greater intracellular macromolecular binding of ⁶³Ni resulting from treatment of cells with ⁶³NiS compared to cultures similarly exposed to ⁶³NiCl₂ paralleled the more potent effects of crystalline NiS in slowing and arresting cell proliferation. Crystalline NiS caused pronounced cell cycle specific blockage at a considerably lower concentration than was required for NiCl₂ to similarly arrest cell growth. Flow cytometry analysis showed that both compounds selectively blocked cell cycle progression in S phase (DNA synthetic stage). These results are suggestive of a common mechanism and site of toxicity inherent to both compounds and related to the cell growth phase during which DNA is replicated.     

Effects of hydrocortisone and aspirin on protein synthesis and post-translational protein modification in cultured cells

Arginyl-tRNA transferase is suggested to function as a post-translational modifier of proteins through the addition of arginine to the NH₂-terminus of specific acceptor proteins. Both hydrocortisone and aspirin produced an age-dependent stimulation of protein synthesis in normal human fibroblasts (IMR90), while producing an inhibition of protein synthesis in SV40 virus-transformed IMR90 cells. The effect of aspirin was confined primarily to the cytoplasmic compartment, whereas hydrocortisone produced its effect at both cytoplasmic and nuclear levels. Neither hydrocortisone nor aspirin had a direct effect on arginyl-tRNA transferase activity in vitro; however, hydrocortisone resulted in a marked increase in the availability of chromosomal proteins subject to modification by arginyl-tRNA transferase. This stimulatory effect was attenuated by increasing culture age. The modified chromosomal proteins were found to be dissociated from native chromatin, suggesting that arginylation either triggered their release or prevented reassociation with chromatin thereafter. Hydrocortisone produced a moderate decrease in the availability of chromosomal proteins for arginylation in SV40 virus-transformed cells, and this effect was not modulated by aging phenomena.     

Reduced cadmium-induced cytotoxicity in cultured liver cells following 5-azacytidine pretreatment

Recent work indicated that administration of the pyrimidine analog 5-azacytidine (AZA), either to cells in culture or to rats, results in an enhancement of expression of the metallothionein (MT) gene. Since MT is thought to play a central role in the detoxification of cadmium, the present study was designed to assess the effect of AZA pretreatment on cadmium cytotoxicity. Cultured rat liver cells (TRL 1215) in log phase of growth were first exposed to AZA (8 microM). Forty-eight hours later, cadmium (10 microM) was added. MT concentrations were then measured 24 hr after the addition of cadmium. A modest increase in MT amounts over control (1.7-fold) was detected after AZA treatment alone. Cadmium alone resulted in a 10-fold increase in MT concentrations. The combination of AZA pretreatment followed by cadmium exposure caused a 23-fold increase in MT concentrations over control. Treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of AZA pretreatment on cadmium induction of MT, indicating that cell division is required. AZA-pretreated cells were also harvested and incubated in suspension with cadmium (250 microM, 37 degrees C) for 0 to 90 min. After incubation intracellular and extracellular fluids were separated by centrifugation through an oil layer. AZA-pretreated cells showed marked reductions in cadmium-induced cytotoxicity as reflected by reduced intracellular potassium loss, glutamic-oxaloacetic transaminase loss, and lipid peroxidation (assessed by thiobarbituric acid reactants) following cadmium exposure. AZA pretreatment had no effect on the cellular uptake of cadmium. Results suggest that AZA pretreatment induces tolerance to cadmium cytotoxicity which appears to be due to an increased capacity to synthesize MT rather than high quantities of preexisting MT at the time of cadmium exposure.     

The cardiotoxicity of hydrogen cyanide as a component of polymer pyrolysis smokes

Groups of adult male rats were repeatedly exposed to the pyrolysis products either of a rigid polyurethane foam or of hemlock, to which was added 0 or 200 ppm hydrogen cyanide under conditions that did not result in depletion of oxygen in the exposure atmosphere. Other groups were exposed to hydrogen cyanide in air or in air plus carbon monoxide. Some of the hydrogen cyanide-exposed groups were pretreated with a chlorpromazine/thiosulfate combination protective against cyanide toxicity. Exposure to hydrogen cyanide without protective pretreatment was associated with mild cardiotoxicity as measured by the magnitude of release of cardiac-specific creatine phosphokinase activity and by the number of ectopic heart beats induced by norepinephrine injection. Much of the difference in the cardiotoxicities of polyurethane and hemlock smokes could be related to their different contents of hydrogen cyanide. However, the prevalence of focal histopathologic lesions in the rat hearts did not correlate well with the other two measures of cardiotoxicity, suggesting that more than one mechanism may be responsible for production of these cardiotoxic effects.