bluuebunny/crossref_metadata_2025 huggingface dataset:
Actions of avermectin B1a on the γ‐aminobutyric AcidA receptor and chloride channels in rat brain
Authors: Abalis Ibrahim M. , Eldefrawi Amira T. , Eldefrawi Mohyee E.
Published: October 2006

The interaction of avermectin B1a (AVM) with the γ‐aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t‐butylbicyclo‐phosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl‐channel blocker 4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl‐influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA‐ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM‐induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA‐receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.
Antagonistic effect of magnesium chloride on the nickel chloride–induced inhibition of DNA replication in chinese hamster ovary cells
Authors: Conway Kathleen , Sen Pramila , Costa Max
Published: October 2006

The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl2) was analyzed by radiolabeled‐thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni2+‐induced inhibition was time‐ and concentration‐ dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl2, which was the highest noncytotoxic concentration utilized. After six hours, 500 μM and 1 mM as well as 2.5 mM NiCl2 all produced the same 50% to 60% reduction in [3H]‐thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 μM or 1 mM NiCl2 treatment began to increase after washout of nickel, but a six‐hour exposure of cells to 2.5 mM NiCl2 produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl2 used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [3H]‐thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl2 for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl2 on DNA replication was prevented by the addition of magnesium chloride (MgCl2) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg2+, since the maximally effective dose of Mg2+ reduced 63Ni2+ uptake by no more than 25% while the inhibition of replication was completely reversed.
The mechanism by which cyclopiazonic acid potentiates accumulation of tetraphenylphosphonium in cultured renal epithelial cells
Authors: Riley Ronald T. , Showker Jency L. , Cole Richard J. , Dorner Joe
Published: October 2006

Cyclopiazonic acid (CPA), a fungal metabolite produced by Aspergillus and Penicillium, potentiated the accumulation of the quaternary cation tetraphenylphosphonium (TPP+) in cultured pig renal epithelial cells. This is the first report of a natural product mediating the tight and apparently nonsaturable binding of a membrane potential probe to subcellular compartments. The potentiated TPP+ accumulation was dose dependent, nonsaturable, and not a result of hyperpolarization across the plasma membrane. Cyclopiazonic acid–potentiated accumulation was completely inhibited by the protonophore carbonylcyanide‐m‐chlorophenylhydrazone (CCCP). Dinitrophenol (DNP), tetrahexylammonium (THA), and n‐ethylmaleimide (NEM) were also effective inhibitors of CPA‐appeared to be energy dependent, TPP+ efflux (in the presence of CCCP) from CPA‐treated cells was incomplete and most of the TPP+ accumulated in the presence of CPA was tightly bound. Dicyclohexylcarbodiimide (DCC), verapamil, and monensin also stimulated TPP+ accumulation, but the TPP+ which accumulated in the presence of these compounds was not tightly bound. As with controls, fractionation of cells which had accumulated TPP+ in the presence of DCC, verapamil, or monensin always resulted in near complete recovery (> 93%) of the TPP+ in the cytosolic fraction, whereas with CPA, greater than 88% of the TPP+ was recovered noncovalently bound in the plasma membrane and mitochondrial fractions. These results are consistent with the hypothesis that CPA‐potentiated TPP+ accumulation is a result of potentiated partitioning of TPP+ into the plasma membranes and mitochondria of LLC‐PK1 cells.
Kinetic parameters of the inhibition of red blood cell aminolevulinic acid dehydratase by triethyl lead and its reversal by dithiothreitol and zinc
Authors: Yagminas Algis P. , Villeneuve David C.
Published: October 2006

In chronic or acute exposure to triethyl lead, a de novo synthesis of aminolevulinic acid dehydratase (δ‐ALAD) in bone marrow and an increased activity in circulating red blood cells can be demonstrated by activating the enzyme with dithiothreitol (DTT) and zinc. We determined the median inhibitory concentration and the apparent inhibition constant for triethyl lead on δ‐ALAD. After dosing with triethyl lead, in vivo inhibition of ALAD only occurred at the high dose, but activation analysis in vitro showed increased ALAD activity to be present at all dose levels in a dose‐dependent fashion. The use of an activation assay for red blood cell ALAD may have value as a bio‐effects monitor of exposure to organic lead.
Mechanism of inhibition of rat brain (Na +‐K +)‐stimulated adenosine triphosphatase reaction by cadmium and methyl mercury
Authors: Ahammadsahib Kabeer I. , Ramamurthi R. , Desaiah Durisala
Published: October 2006

The mechanisms of inhibition of rat brain Na +‐K +‐ ATPase by cadmium chloride (CdCl2) and methylmercuric chloride (CH3HgCl) were studied in vitro by assessing the effects of these heavy metals on this enzyme and associated component parameters. Both the heavy metals significantly inhibited the overall Na +‐K + ‐ATPase in a concentration‐dependent manner with an estimated median inhibitory concentration (IC‐50) of 3.2 × 10−5M for CdCl2 and 6 × 10−6M for CH3HgCl. Protection of enzyme against heavy metal inhibition by 5 × 10−5M to 1 × 10−4 M dithiothreitol (DTT) and glutathione (GSH) or cysteine (CST) indicates that both monothiols and dithiols have the same ability in regenerating sulfhydryl (–SH) groups or chelating the metals. Inhibition of K+‐p‐nitrophenyl phosphatase (K+‐PNPPase), the component enzyme catalyzing the K+‐dependent dephosphorylation in the overall Na +‐K +ATPase reaction by these heavy metals, indicates that the mechanism of inhibition involves binding to this phosphatase. Reversal of K+‐PNPPase inhibition by DTT, GSH, and CST suggests sulfhydryl groups as binding sites. Binding of 3H‐oubain, a cardiac glycocide and inhibitor of both phosphorylation and dephosphorylation, to brain fraction was significantly decreased by CH3HgCl, and this inhibition was reversed by the three thiol compounds, suggesting presence of –SH group(s) in the ouabain receptor site. Cadmium chloride failed to inhibit the binding of this receptor, indicating that the mechanics of inhibition of ATPase by CH3HgCl and CdCl2 are different from each other. The results suggest that the critical conformational property of enzyme common to both kinase (E1) and phosphatase (E2) is susceptible to CH3HgCl whereas only phosphatase is sensitive to CdCl2.
Exposure to different forms of cadmium in mice: Differences in metallothionein and alphafetoprotein mRNA induction in liver and kidney
Authors: Koropatnick James , Cherian M. George
Published: October 2006

Cadmium (Cd) exposure in mice induces transcription of metallothionein (MT) mRNA and protein accumulation in both liver and kidney. Resistance to hepatotoxicity through chronic exposure to heavy metals is the result of this induction. However, the same chronic exposure results in damage to kidney. We report here that acute exposure of mice to Cd as cadmium sulfate (CdSO4), which resulted in preferential accumulation of metal in liver, or Cd‐metallothionein (CdMT), which resulted in preferential metal accumulation in kidney, induced MT mRNA accumulation in both liver and kidney. However, MT mRNA accumulated to a level twofold higher in liver than in kidney in response to CdSO4. Equivalent doses of CdMT induced MT mRNA accumulation to an equal degree in kidney and liver. While MT mRNA accumulation in kidney was directly proportional to the amount of cadmium in the organ, this was not the case in liver. There, liver MT mRNA was elevated in the absence of elevated tissue cadmium levels. Interestingly, CdMT induced alphafetal protein (AFP) mRNA accumulation in kidney, but not liver. It appears that (a) maximal MT mRNA accumulation in kidney is less than in liver, and (b) liver is capable of accumulating MT mRNA in response to even very low cadmium exposure that may not result in elevated tissue cadmium.
Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine receptors
Authors: Bakry Nabil M. S. , El‐Rashidy Amina H. , Eldefrawi Amira T. , Eldefrawi Mohyee E.
Published: October 2006

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti‐ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E‐115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC‐50) of ACh‐esterase activity. However, a small population of m‐ACh receptors had an affinity as high as that of ACh‐esterase for the OP compound. This population is identified by its high‐affinity [3H]‐cis‐methyldioxolane ([3H]‐CD) binding. Although sarin, soman, and tabun had no effect, (O‐ethyl S[2‐(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitivel the binding of receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50‐fold more potent in inhibiting carbamylcholine (carb)‐stimulated [3H]‐cGMP synthesis in N1E‐115 neuroblastoma cells—both acting as m receptor antagonist. All five OPs inhibited [3H]‐CD binding, with IC‐50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n‐ACh receptor (identified by inhibition of [3H]‐phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [125I]‐α‐bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n‐ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n‐ACh as well as m‐ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh‐esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m‐ACh receptors, which have a high affinity for CD. Such effects on m‐ACh receptors may play an important role in the toxicity of certain OP compounds.
Chlordecone is a potent in vitro inhibitor of oligomycin‐insensitive mg2+ ‐ATPase of rat bile canaliculi–enriched fraction
Authors: Curtis Lawrence R.
Published: October 2006

The oligomycin‐insensitive Mg2+ ‐ATPase (OIMATPase) of rat bile canaliculi–enriched fraction (BCEF) was inhibited by chlordecone (CD) in vitro (IC‐50 = 25 μM). Kinetic analysis indicated noncompetitive inhibition. Inhibition of OIMATPase by filipin but not by atractyloside verified plasma membrane origin of activity. The cholestatic agents α‐naphthyl isothiocyanate (ANIT) and taurolithocholate (TLC) decreased OIMATPase activity at in vitro concentrations of 33 and 162 μM, while taurocholate (a choleretic bile salt), ethynylestradiol, and manganese did not. Cholestatic drugs with primary intracellular sites of action (colchicine and phalloidin) were ineffective OIMATPase inhibitors in this concentration range. Inhibition of OIMATPase by N‐ethylmaleimide (NEM) and dicyclo‐hexylcarbodiimide (DCCD) indicated some H+ ‐ATPase activity in BCEF. In vitro sensitivity of OIMATPase of BCEF to CD, ANIT, and TLC suggested the bile canaliculus as a subcellular‐level target for their cholestatic actions.
Genetically engineered V79 chinese hamster cell expression of purified cytochrome P‐450iib1 monooxygenase activity
Authors: Platt K. L. , Molitor E. , Döhmer J. , Dogra S. , Oesch F.
Published: October 2006

Chinese hamster V79 fibroblasts, frequently used as target cells in short‐term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA‐encoding rat liver cytochrome P‐450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so‐called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16α‐ and 16β‐hydroxy‐T as well as 4‐androsten‐3,17‐dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio‐ and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P‐450IIB1 in a reconstituted system.
Mutagenicity of tetrachloroethene in the ames test—metabolic activation by conjugation with glutathione
Authors: Vamvakas Spyridon , Herkenhoff Martin , Dekant Wolfgang , Henschler Dietrich
Published: October 2006

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S‐(1,2,2‐trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of γ‐glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S‐transferases in presence of GSH and rat kidney fractions resulted in a time‐dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the β‐lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by β‐lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.
Effect of verapamil on glycogenolysis and gluconeogenesis in the perfused rat liver
Authors: Badr Mostafa
Published: October 2006

In perfused livers from fed rats, rates of glucose production (glycogenolysis) were 133 ± 12 μmol/g/hr. Infusion of 2 μM verapamil into these livers decreased the rates of glucose production significantly to 97 ± 15 μmol/g/hr within 10 min. Conversely, rates of production of lactate plus pyruvate (glycolysis) of 64 ± 6 μmol/g/hr were not significantly altered by verapamil (60 ± 3 μmol/g/hr). When 50 μM verapamil was infused, however, rates of both glycogenolysis and glycolysis were diminished to 56 ± 11 and 43 ± 5 μmol/g/hr, respectively. In perfused livers from fasted rats, infusion of 20 mM fructose increased the rates of production of glucose (gluconeogenesis) significantly from 11 ± 7 to 121 ± 17 μmol/g/hr. These rates reached 138 ± 7 μmol/g/hr upon the simultaneous infusion of verapamil (2 μM). In these livers, fructose also increased rates of production of lactate from 6 ± 2 to 132 ± 11 μmol/g/hr, which were further increased to 143 ± 8 μmol/g/hr when 2 μM verapamil was infused. The results show that calcium‐dependent processes involved in hepatic carbohydrate metabolism respond differently to the calcium channel blocker verapamil. Low concentrations of verapamil inhibited glycogenolysis significantly while having no effect on either glycolysis or gluconeogenesis. These data suggest that these two processes have different sensitivities to changes in intracellular calcium concentrations and/or different sources of regulatory calcium.
The interaction of phenyldichloroarsine with erythrocytes
Authors: Chong Steven , Dill Kilian , McGown Evelyn
Published: October 2006

The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U‐14Clphenyldichloroarsine (PDA)] in phosphate‐buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02–0.3 μmol PDA/109 cells, depending on the species. Saturation points correlated well with each respective species' erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 μmol/109 cells. Hemolysates of PDA‐treated erythrocytes were subjected to Sephadex G‐75 gel filtration chromatography. 14C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione‐containing) fractions. In all other species, the 14C eluted almost exclusively with the glutathione‐containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDA/mol with Kd ∼ 5 μM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.
Patulin‐induced ion flux in cultured renal cells and reversal by dithiothreitol and glutathione: A scanning electron microscopy (sem) X‐ray microanalysis study
Authors: Hinton Dorothy M. , Riley Ronald T. , Showker Jency L. , Rigsby William E.
Published: October 2006

Patulin (PAT), a compound produced by certain species of Aspergillus, Penicillium, and Byssochlamys, is frequently found associated with agricultural commodities. PAT has many effects on membrane function, including the inhibition of the isolated Na+‐K+ ATPase. In this study, a scanning electron microscope equipped with an energy dispersive spectroscopy X‐ray microanalysis system was used to examine individual cultured renal epithelial cells (LLC‐PK1) in order to determine the effects of PAT on the relative intracellular ion concentrations. The estimated EC50 (60 min) for both sodium influx and potassium efflux was between 10 and 50 μm for ouabain. For PAT, the EC50 (60 min) was 250 μm for sodium influx and 100 μm for potassium efflux. However, 1 mM patulin at 240 min caused complete reversal of the sodium and potassium content of cells, and 1 mM ouabain at 240 min did not. The effect of patulin on sodium and potassium flux was both concentration and time dependent and was reversed by dithiothreitol and glutathione. PAT (250 μM) but not ouabain (250 μM) induced massive blebbing of LLC‐PK1 cells. Thus, the interaction of PAT with cellular membranes involves both alterations in the regulation of intracellular ion content and the cyto‐skeleton. We hypothesize that patulin alters intracellular ion content via Na+–K+ ATPase and non‐Na+–K+ ATPase mechanisms.
Distribution and peroxidative oxidation of 2‐t‐butyl‐4‐methoxyphenol in rat tissues after a single intraperitoneal dose
Authors: Della Corte Laura , Bianchi Loria , Valoti Massimo , Sgaragli Giampietro
Published: October 2006

The distribution of 2‐t‐butyl‐4‐methoxyphenol (BHA) and its conversion to 2,2′‐dihydroxy‐3,3′‐di‐t‐butyl‐5,5′‐dimethoxydiphenyl (di‐BHA) in rat tissues at different times (1–96 hr) following the intraperitoneal administration of a single dose of BHA (32 mg kg−1 body weight) were monitored by gas chromatography‐mass spectrometry (GC/MS) analysis of both compounds. High BHA levels were found in the intestine and liver persisting up to 24 hours (5.5–20.7 and 1.8–3.3 μg g−1 wet weight, respectively). In these tissues, values of the area under the experimental concentration curve (AUC0–24) were 285 and 49 times higher, respectively, than those observed in plasma (945 ng mL−1 hr), AUC0–24 values in kidney, spleen, erythrocytes, and brain were 2–7 times higher, whereas values below those found in plasma were observed in lung and muscle. The metabolite di‐BHA could be detected in the intestine, kidney, and spleen, amounting to 5–8% of BHA. These findings indicate that rat intestine is capable of transforming in vivo BHA into di‐BHA even when the former compound is administered intraperitoneally and that this capacity is shared by the kidney and spleen.
Inhibition of soybean lipoxygenase and mouse skin tumor promotion by onion and garlic components
Authors: Belman Sidney , Solomon Jerome , Segal Alvin , Block Eric , Barany George
Published: October 2006

Onion and garlic essential oils were previously shown to inhibit mouse skin tumor promotion, as were the enzymes, lipoxygenase, and cyclooxygenase. In the present study, the inhibition of soybean lipoxygenase (EC 1.13.11.12) by onion and garlic components and related compounds was investigated. The IC50values as well as the kinetic inhibition constants were determined for the most active compounds. Di‐(1‐propenyl) sulfide, an analog of the substrate moiety required for oxygenase action, was the only irreversible inhibitor observed withKi=59 μM andk3=0.53/min. Inhibition in the presence of substrate was uncompetitive at 88 and 132 μM linoleic acid withKi=129 μM. At 173 μM linoleic acid, however, inhibition was competitive withKi=66 μM. Dially trisulfide, allyl methyl trisulfide, and diallyl disulfide were competitive inhibitors, while 1‐propenylpropyl sulfide and (E, Z)‐4,5,9‐trithiadodeca‐1,6,11‐triene 9‐oxide (ajoene) were mixed inhibitors. Nordihydroguaiaretic acid (NDGA), the most potent lipoxygenase inhibitor, was a competitive inhibitor withKi=0.29 μM. The results indicate a relative potency of inhibition for structural features in the following order: di(1‐propenyl) sulfide > an alkenyl trisulfide > an alkenyl disulfide. Di(n‐propyl) disulfide, a major onion oil component, inhibited neither lipoxygenase nor promotion. Di(1‐propenyl) sulfide and ajoene inhibited both. This suggests that the inhibition of lipoxygenase may be involved in anti‐promotion.
Effects of TCDD on the EGF receptor of XB mouse keratinizing epithelial cells
Authors: Kawamoto Toshio , Matsumura Fumio , Madhukar B. V. , Bombick David W.
Published: October 2006

TCDD was found to cause a marked inhibition of 125I‐epidermal growth factor (EGF) binding to its receptor on the cell surface of XB mouse keratinizing epithelial cells (XB cells) cultured in vitro. The EC50 concentration was estimated to be on the order of 3 × 10−11 M 24 hours after TCDD administration. As early as 12 hours after the addition of 10−9 M of TCDD, XB cells showed signs of a decline in 125I‐EGF binding levels. The level of such EGF receptor downregulation reached a maximum at 24 hours, continued until day 2, but completely recovered by day 3. This was accompanied by a rise in protein kinase activities, particularly those of the protein tyrosine kinases during the initial period of 6–24 hours. To test the hypothesis that the EGF receptors of the cells, by showing TCDD‐induced symptoms of downregulation, actually are being activated and triggering EGF‐like signals, we examined the effects of both TCDD and exogenously added EGF on cell morphology, colony formation degree of keratinization, the pattern of activation of protein kinases and de novo protein synthesis, and EGF receptor phosphorylation. Based on the similarity of cell responses to these between TCDD‐ and EGF‐treated cells, we concluded that TCDD, directly or indirectly, causes activation of the EGF receptor. In contrast, 12‐O‐tetradencanoylphorbol‐13‐acetate (TPA), which is known to downregulate EGF receptors by blocking their protein tyrosine kinase, produced dissimilar end results. The balance of evidence support the notion that the action of TCDD in this cell line is tightly coupled to the activation of the EGF receptor and that one of the key consequences of such a biochemical change is that it signals these cells to commit to terminal differentiation.
Competitive partial inhibitors of serum albumin‐catalyzed sulfur cyanolysis
Authors: Jarabak Rebecca , Westley John
Published: October 2006

Efforts to locate the active site for sulfur cyanolysis catalyzed by bovine serum albumin have led to systematic tests of several compounds that inhibit the catalyzed reaction. Hexanoate and 5‐dimethylaminonaphthalene‐1‐sulfonate bind at the same site and are partial inhibitors competitive with cyanide, uncompetitive with respect to sulfur. Various dansyl amino acids and 1‐anilino‐8‐naphthalene sulfonate display the same inhibitory behavior but bis (1‐anilino‐8‐naphthalene sulfonate) is a total inhibitor competitive with cyanide. These findings are interpreted to indicate that the cyanolysis active site is near, but not at, one of the short‐chain fatty acid binding sites on albumin subdomain 2‐AB or 3‐AB. Both ionic repulsion and steric considerations are implicated in the mechanisms of inhibition.
Perturbations in polyamines and related enzymes following chlordecone‐potentiated bromotrichloromethane hepatotoxicity
Authors: Rao Shrinivas B. , Young Robert A. , Mehendale Harihara M.
Published: October 2006

The mechanism by which chlordecone (CD) amplifies the hepatotoxicity of halomethanes such as CCl4, CHCl3, and BrCCl3 has been a subject of intense study. Recent work has shown that suppression of hepatocellular regeneration leads to accelerated progression of liver injury leading to complete hepatic failure due to an unusual interaction between individually nontoxic low‐dose combination of CD and CCl4. Since polyamines are involved in cell division, their levels reflect the extent to which there is suppression of hepatocellular regeneration during CD and CCl4 interaction. The present studies were designed to investigate the polyamine levels and associated enzymes in livers of rats treated with BrCCl3 alone or CD and BrCCl3 low‐dose combination in order to confirm whether the sequence of events of hepatotoxicity is similar to that seen in CCl4 toxicity or that seen during CD and CCl4 interaction. The extent of liver toxicity in rats fed 10 ppm chlordecone (CD) for 15 days prior to the injection of a single low dose of BrCCl3 (15 μL/kg body weight) or after exposure to a high dose of BrCCl3 (80 μL/kg body weight) without CD pretreatment, was similar 6 and 24 hr later as assessed by plasma transaminase levels. There was also an increase in transaminase levels, in rats exposed to a single low dose of BrCCl3 alone (15 μL/kg body weight) but this increase was far below the high‐dose exposure alone or the combination treatment. Hepatic levels of ornithine decarboxylase, S‐adeno‐sylmethionine decarboxylase, N1‐acetylputrescine, N1‐acetylspermidine, putrescine, spermidine, and spermine at the end of 24 hr increased after exposure to a low dose of BrCCl3 alone as compared to exposure to a high dose alone or the low‐dose combination of CD and BrCCl3. Liver spermidine N1‐acetyltrans‐ferase was elevated at 2, 6, and 24 hr after exposure to a high dose of BrCCl3 alone as compared to treatment with a low‐dose combination of CD and BrCCl3 suggesting decreased synthesis of this enzyme, in spite of a greater need as seen from liver transaminase levels. In general, it was observed that there is significant elevation in some polyamines and related enzymes during toxicity of a low dose of BrCCl3 which seemed to stabilize within 24 hr. This was not observed with the other two groups of rats exposed either to BrCCl3 high dose alone or the low‐dose combination of CD and BrCCl3. Results indicate that CD and BrCCl3 low‐dose combination treatment causes increased liver toxicity resulting in compromised polyamine metabolism which is coincidental with suppressed hepatocellular regeneration leading to accelerated progressive phase of liver injury culminating in complete hepatic failure. These findings point to the possibility that the mechanism of potenti‐ation of BrCCl3 hepatotoxicity by CD is similar to that seen for CD and CCl4 interaction.
Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by aroclor 1254 or phenobarbital
Authors: Haake‐McMillan J. M. , Safe S. H.
Published: October 2006

The constitutive and Aroclor 1254‐in‐duced activities of hepatic microsomal benzo[a]‐pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11‐day‐old noninduced male rats, benzo[a]pyrenediones and 9‐hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21‐day‐old males benzo[a]pyrene‐diones and benzo[a]pyrene‐9,10‐dihydrodiol were predominant. In 60‐ and 120‐day‐old animals 3‐hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5‐di‐hydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21‐day‐old immature male rats, in which there was a 330‐ and 4.5‐fold increase in the formation of 3‐hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3‐ to 10.1‐fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 μmol/kg) or Aroclor 1254 (100 μmol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120‐day‐old animals. Neonatal exposure to phenobarbital resulted in a decrease in the Aroclor 1254‐induced rates of formation of dihydrodiol and quinone benzo[a]pyrene metabolites in male rat microsomes, an increase in the rates formation of the 9,10‐ and 7,8‐dihydrodiols in Aroclor 1254‐induced female rat microsomes, and a decrease in the constitutive and Aroclor 1254‐induced 3‐ and 9‐hydroxylase activities in hepatic microsomes from either sex. Neonatal exposure to Aroclor 1254 (100 μmol/kg) resulted in significantly lower Aroclor 1254‐induced rates of formation of the 9,10‐, 4,5‐ and 7,8‐dihydrodiol, quinone, and 9‐hydroxybenzo[a]pyrene metabolites in 120‐day‐old male rat microsomes and significantly decreased rates of formation of the 4,5‐dihydrodiol and quinone benzo[a]pyrene metabolites in Aroclor 1254‐induced female rat hepatic microsomes. Only minor differences were observed in the constitutive benzo[a]pyrene hydroxylase activities in microsomes from male or female rats neonatally treated with either phenobarbital or Aroclor 1254. These results demonstrate that neonatal exposure to phenobarbital or Aroclor 1254 can alter the adult metabolism of xenobiotics such as benzo[a]pyrene.
Linoleic acid‐induced endothelial cell injury: Role of membrane‐bound enzyme activities and lipid oxidation
Authors: Ramasamy Santhini , Boissonneault Gilbert A. , Decker Eric A. , Hennig Bernhard
Published: October 2006

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane‐bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 μM. fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 μM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane‐bound enzymes, angiotensin‐converting enzyme (ACE), and Ca2+‐ATPase increased steadily with increasing time of cell exposure to 90 μM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 μM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [3H]‐adenine. Incubation of 18:2‐supplemented serum‐containing culture media with 1000 μM 18:2 at 37°C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.
Effects of isoflurane on the Dnase I activity in an isolated enzyme preparation and on the Dnase I‐G actin complex
Authors: Reitz Manfred , Von Pfeil Andrea , Hundt Ferdinand , Lanz Egon
Published: October 2006

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and in the DNase I‐globular (G) actin complex were investigated. DNase I, DNase I‐G actin complex, and G actin were exposed to various (0.2–4.0 vol%) isoflurane concentrations for 180 min. Thereafter, DNase I activity was determined. DNase I activity was inhibited in relation to time and concentration of isoflurane exposure. At concentrations ranging from 0.2 to 1.0 vol% of isoflurane inactive DNase I was activated in the DNase I‐G actin complex. The DNase I inhibitor G actin showed a reduced capability to inhibit DNase I following isoflurane exposure. Albumin can inhibit the DNase I inactivation possibly by competition in the reactions between DNase I/albumin and isoflurane. After exposure to isoflurane the absorption maximum of DNase I was identical with the absorption maximum of heat‐denaturated DNase I. The results suggest a mechanism by which isoflurane may affect DNA in an indirect way at concentrations to which the patient is exposed during clinical anesthesia.
Association of microcystin‐LR and its biotransformation product with a hepatic‐cytosolic protein
Authors: Robinson Nancy A. , Matson Charles F. , Pace Judith G.
Published: October 2006

Microcystin‐LR (MCYST‐LR), a cyclic peptide hepatotoxin, associates with high‐molecular‐weight, liver cytosolic components. Repetitive cycles of heat denaturation and pronase digestion released 80 ± 6% of the bound radiolabel from these components, parent toxin (22%), and two biotransformation products, with high‐performance liquid chromatography (HPLC) retention times of 6.7 (52%) and 5.6 (13%) min. Both parent and the biotransformed (6.7 min) toxin appeared to be covalently bound to a monomeric protein of molecular weight 40,000 (protein plus radiolabeled toxin). Binding and biotransformation reactions were time‐ and temperature‐dependent and did not require endogenous molecules <6,000 daltons. The binding appeared to be saturable with a maximum of 20 pmol MCYST‐LR bound per mg protein. The binding protein(s) and biotransformation activity were present in rat liver, brain, kidney, heart, lung, small intestine, large intestine, testes, skeletal muscle, and to a lesser extent, in fat. Okadaic acid, a specific protein phosphatase inhibitor, showed a concentration‐dependent inhibition of [3H]MCYST‐LR binding to hepatic cytosol. The molecular weight and organ distribution of the binding protein(s), and inhibition of binding by okadaic acid were consistent with one of the binding sites being the catalytic subunit of protein phosphatase type 2A.
Role of cytochrome P‐450 in ochratoxin a‐stimulated lipid peroxidation
Authors: Omar Rabeea F. , Rahimtula Anver D. , Bartsch Helmut
Published: October 2006

The role of cytochrome P‐450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH‐cytochrome P‐450 reductase, Fe3+, ethylenediaminetetra‐acetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P‐450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe3+. Cobalt protoporphyrin is known to cause an extensive and long‐lasting depletion of hepatic cytochrome P‐450 in rats, and it has been used to evaluate the role of hepatic cytochrome P‐450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin‐pretreated rats underwent ochratoxin A‐dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin‐pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P‐450 in ochratoxin A‐induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A‐induced lipid peroxidation.
Acrylonitrile interaction with testicular DNA in rats
Authors: Ahmed Ahmed E. , Abdel‐Rahman Sherif Z. , Nour Deen Amr M. ‐Al
Published: October 2006

In the present study we report the in vivo interaction of acrylonitrile (VCN) with testicular tissue in rats. Covalent binding of radioactivity to testicular tissue DNA was examined for a period of 72 hr after a single oral dose (46.5 mg/kg) of [2, 3‐14C] VCN. Maximal covalent binding was observed at 0.5 hr (8.9 μmol VCN equivalent/mol nucleotide). Binding decreased gradually thereafter but was still detected (2.5 μmol VCN equivalent/mol nucleotide) at 72 hr following VCN administration. Further, we examined the effects of VCN on DNA synthesis and repair in the testes of rats following a single oral dose (46.5 mg/kg) of VCN to clarify the impact of the covalent binding observed on the testicular genetic material. A significant decrease in DNA synthesis (80% of control) was observed at 0.5 hr after treatment. At 24 hr following acrylonitrile administration, testicular DNA synthesis was severely inhibited (38% of control). Testicular DNA repair was increased 1.5‐fold at 0.5 hr and more than 3.3‐fold at 24 hr following treatment with VCN. These results suggest that VCN can act as a multipotent genotoxic agent by alkylating DNA in testicular tissue and may affect the male reproductive function by interfering with testicular DNA synthesis and repair processes.
Lack of evidence for perfluorodecanoyl‐ or perfluorooctanoyl‐coenzyme a formation in male and female rats
Authors: Kuslikis Benedict I. , Vanden Heuvel John P. , Peterson Richard E.
Published: October 2006

Perfluorodecanoic (PFDA) and perfluorooctanoic (PFOA) acids belong to the structurally diverse group of compounds known to cause peroxisomal proliferation. It has been hypothesized that the common mode of action of these compounds is that they act through an activated coenzyme A (CoA) thioester. Using rat liver microsomal and isolated rat hepatocyte incubation conditions that were effective in producing a COA conjugate of clofibric acid, no corresponding COA derivative could be found for either PFDA or PFOA.
Effect of endrin on the hepatic distribution of iron and calcium in female sprague‐dawley rats
Authors: Bagchi D. , Bagchi M. , Hassoun E. , Stohs S. A.
Published: October 2006

The distribution of iron and calcium in hepatic subcellular fractions of female rats treated with endrin (1, 2, 3, 4, 10, 10‐hexachloro‐6, 7‐epoxy‐1, 4, 4α, 5, 6, 7, 8, 8α‐octahydroendo, endo‐1, 4:5, 8‐dimethanonaphthalene) was determined. Endrin in corn oil was administered orally to rats in single doses of 3, 4.5, or 6 mg/kg, and the animals were killed at 0, 12, 24, 48, or 72 hr posttreatment. Iron and calcium were determined by atomic absorption spectroscopy. The administration of endrin increased the iron content of mitochondria and decreased the iron content of microsomes and nuclei. Significant increases occurred in the calcium content of mitochondria, microsomes, and nuclei. Thus, the results indicate that with respect to the subcellular distribution of iron and calcium, endrin produces differential effects. Vitamin E succinate administration partially prevented the endrin‐induced hepatic alterations in iron and calcium homeostasis. Endrin also produced dose‐ and time‐dependent increases in the liver and spleen weight/body weight ratios, while decreasing the thymus weight/body weight ratios. The altered distribution of calcium and iron may contribute to the broad range of effects of endrin.
Modifiers of mercaptopyruvate sulfurtransferase catalyzed conversion of cyanide to thiocyanate in vitro
Authors: Wing David A. , Baskin Steven I.
Published: October 2006

The enzyme mercaptopyruvate sulfurtransferase appears to play an important role in the in vivo detoxification of cyanide. It does so by transferring sulfur to cyanide to produce thiocyanate, which is less toxic and may be excreted through the kidney. Several compounds were tested for their ability to affect the rate of enzyme catalyzed thiocyanate formation in vitro. The studies were carried out using both a partially purified bovine kidney extract and a highly purified enzyme preparation. Hypotaurine and methanesulfinic acid doubled sulfurtransferase activity in the partially purified extract at 30 mM, but inhibited the purified enzyme to 57% (hypotaurine) and 27% (methanesulfinic acid) of control activity at the same concentration. Pyruvate, phenylpyruvate, oxobutyrate, and oxoglutarate each inhibited the extract and purified forms of mercaptopyruvate sulfurtransferase. Phenylpyruvate was the most effective inhibitor, reducing activity to 0.2% of control values in the extract, and 11% of control values for purified MPST when added to the reaction at 30 mM. Other compounds tested (see Table 1) had a negligible effect on sulfurtransferase activity. A heat stable cofactor was found in boiled kidney extract which stimulated sulfurtransferase activity in the extract but inhibited sulfurtransferase activity in the purified enzyme, as was observed for hypotaurine and methanesulfinate. The boiled extract had no thiocyanate forming activity of its own. The cofactor operated in synergy with methanesulfinate, but independently of hypotaurine.
Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells
Authors: Abdallah Elsayed A. M. , Jett David A. , Eldefrawi Mohyee E. , Eldefrawi Amira T.
Published: October 2006

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor‐coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3‐specific antagonist 4‐DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4‐DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems.

Paraoxon (100 μM) reduced Bmax of [3H]4‐DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β‐adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol‐induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC‐stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M3 muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the Gi proteinadenylyl cyclase system, and produces CBC‐like action, that is, inhibition of the forskolin‐stimulated [3H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.
The role of calcium ions for the expression of ricin toxicity in cultured macrophages
Authors: Naseem Syed M. , Wellner Robert B. , Pace Judith G.
Published: October 2006

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited 3H‐leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca2+‐free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg2+ did not affect protein synthesis or binding of 125I‐ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.