bluuebunny/crossref_metadata_2025 huggingface dataset:
Embryotoxicity induced by diethylstilbestrol in vitro1
Authors: Greenaway J. C. , Fantel A. G. , Beyer B. K. , Juchau M. R.
Published: October 2006

The embryotoxic potential of diethylstilbestrol (DES) was examined in a whole embryo culture system containing a P‐450–dependent bioactivating system. Sprague‐Dawley rat embryos were explanted on day 10 and cultured for 24 hours. Concentration‐dependent effects of DES on embryonic growth parameters, viability, and embryotoxicity were observed. Concentrations of DES greater than 0.26 mM (final concentration) produced 100% embryolethality, while those below 0.15 mM were without significant effects. At a final concentration of 0.19 mM, DES produced only a slight increase in embryolethality. The same concentration elicited a marked increase in observed embryotoxicity, including prosencephalic hypoplasia, incomplete axial rotation, and open neural tubes. In addition, reductions in embryonic length, somite number, and protein and DNA content were observed. An exogenous P‐450–dependent hepatic biotransforming (catechol‐generating) system failed to alter either the incidence of observed toxic effects or measured growth parameters. Likewise, exposure of cultured embryos to 20% carbon monoxide (CO) failed to reduce DES‐induced embryotoxicity, indicating a lack of participation of an endogenous P‐450‐dependent embryonic bioactivating system. Arachidonic acid (0.20 mM) and/or indomethacin (0.50 mM) also had no observable effect on DES‐induced embryotoxicity, suggesting that prostaglandin synthase was not involved in the embryotoxic activity of DES, as has been proposed to explain its carcinogenic effect. The antioxidants N‐acetylcysteine (1.14 mM) and α‐tocopherol (0.08 mM) failed to protect against DES‐induced embryotoxicity, while the antiestrogen tamoxifen (up to 0.85 mM) actually enhanced this effect of DES in culture. The DES analogs Z,Z‐dienestrol (DIES, 0.10 mM) and hexestrol (HES, 0.48 mM) were both embryotoxic in vitro. The presence of an exogenous P‐450‐dependent hepatic biotransforming system appeared to protect against HES‐induced embryolethality but had little effect upon DIES‐induced embryotoxicity. The results were consistent with a direct effect of DES independent of either estrogenicity or exogenously generated metabolites.
Significance of mirex‐caused hypoglycemia and hyperlipidemia in rats
Authors: Jovanovich Lela , Levin Sally , Khan M. A. Q.
Published: October 2006

Treatment of rats with mirex (40 ppm in diet) caused hypoglycemia, liver enlargement, and inhibition of adrenal corticosteroid‐synthesizing enzyme activity. At toxic dosages (20,000 ppm mirex in diet, which has a lethal toxicity‐50 [LT‐50] of ten days) poisoned female rats showed severe hypoglycemia, fatty liver, adrenal hyperplasia, hypophagia, lipid mobilization, and body weight (bw) loss. A 50 μg/kg intraperitoneal (IP) dose of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) in male rats caused similar effects two days posttreatment. Hypoglycemia could be overcome by prednisone (which also inhibited adrenocorticoid‐synthesizing enzyme activities) but not by streptozotocin treatment, indicating that hypoglycemia may be related to glucocorticoid deficiency resulting from inhibition of their synthesis and not by direct effects on pancreatic β‐cells. Glucocorticoid deficiency could also cause increased release of adrenocorticoid hormone (ACTH), which may enhance fat mobilization caused by hypophagia.
Allylisopropylacetamide induces rat hepatic ornithine decarboxylase
Authors: Kitchin Kirk T. , Brown Janice L.
Published: October 2006

In rat liver, allylisopropylacetamide (AIA) treatment strongly induced (25‐fold) the activity of rat hepatic ornithine decarboxylase (ODC). By either the oral or the subcutaneous route, AIA produced a long‐lasting induction (30 to 40 hours) of hepatic ODC activity. Three analogs of AIA, propylisopropylacetamide (PIA), allobarbital, and allylbenzene, were active ODC inducers while a fourth, allylacetate, was not. Although induction of hepatic aminolevulinic acid (ALA) synthetase activity and the accumulation of hepatic porphyrins depend on the allyl moiety of AIA, this is not the case with hepatic ODC induction. Allylisopropylacetamide did not elevate serum alanine aminotransferase (SGPT) nor did it cause DNA damage, as measured by the alkaline elution assay. Thus, hepatic cell death is not a likely explanation of AIA's long‐lasting induction of ODC. As AIA does not belong to any of the common categories of ODC inducers, it may be the chemical prototype of a new class of hepatic ODC inducers.
Effects of metal cations on pituitary hormone secretion in vitro
Authors: Cooper Ralph L. , Goldman Jerome M. , Rehnberg Georgia L. , McElroy W. Keith , Hein Joy F.
Published: October 2006

Increased body burdens of metal cations are known to affect adversely reproductive function in several species. The effects of these metals on gonadal function are well documented. In contrast, little is known about their possible direct effects on pituitary hormone release. The purpose of this study was to determine, in vitro, the effects of nickel, cadmium, and zinc (50 μM) on both baseline and potassium chloride (KCl)‐stimulated pituitary luteinizing hormone (LH), prolactin (Prl), and thyroid‐stimulating hormone (TSH) release. Anterior pituitary fragments from adult male Long‐Evans rats were evaluated using a continuous‐flow perifusion system. Baseline and stimulated LH releases were unaffected by nickel and zinc; however, cadmium caused an increase in baseline LH secretion. Baseline Prl release was decreased by zinc, while cadmium resulted in increased release of this hormone. Stimulated Prl release was lower during exposure to zinc but unaltered by nickel and cadmium. Following exposure to zinc, a rebound in stimulated release was noted for all three hormones measured. These results showed that the metal cations tested did have a direct effect on pituitary hormone release at a dose lower than those reported to alter testicular function in vitro. Furthermore, the changes in pituitary hormone secretion varied depending upon the metal and hormone being evaluated.
Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate
Authors: Eldefrawi Mohyee E. , Schweizer Gordon , Bakry Nabil M. , Valdes James J.
Published: October 2006

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [3H]‐phencyclidine ([3H]‐PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [3H]‐ACh or 125I‐α‐bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time‐dependent manner [3H]‐PCP binding to the receptor's high‐affinity allosteric site, it inhibited the ACh or carbamylcholine‐stimulated [3H]‐PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo‐first‐order rate of [3H]‐PCP binding to that of an agonist‐desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high‐affinity noncompetitive sites.
Investigations on the role of free radical processes in hexachlorobenzene‐induced porphyria in mice
Authors: Rizzardini M. , Graziani A. , Carugo C. , Cantoni L.
Published: October 2006

Male C57BY/10 mice were chronically fed hexachlorobenzene (HCB) (0.02% of the diet) alone or in combination with a single subcutaneous dose of iron (12.5 mg iron per mouse). After eight weeks the group of mice pretreated with the iron overload was highly sensitized to the porphyrogenic effect of HCB, as shown by liver porphyrin accumulation. A synergistic effect of iron was evident on other parameters too, such as HCB‐induced hepatic damage, activation of type O of xanthine oxidase, and decreased activity of copper zinc superoxide dismutase and glutathione peroxidase(s). None of these parameters was affected by iron alone.

Iron alone and in association with HCB markedly raised the level of lipid peroxides, the increase in the HCB group being smaller. The combined treatment resulted in a significant reduction of HCB's inductive effects on microsomal heme and cytochromes P‐450 and b5 and on the activity of aryl hydrocarbon hydroxylase. The content of nonprotein sulfhydryl groups was reduced to the same extent in mice treated with HCB or HCB plus iron.

The results suggest that reactive intermediates such as are formed by lipid peroxidation are not sufficient on their own to create the conditions for uroporphyrinogen decarboxylase impairment, as evident in the group of mice receiving iron overload alone. Conversely, HCB administration induced a specific condition of imbalance in the liver between formation and inactivation of reactive intermediates which was associated with hepatic porphyrin accumulation and was potentiated by concomitant administration of iron.
Cyclosporin‐mediated increase in kidney glutathione and effects on γ‐glutamyl‐cycle enzymes
Authors: Mayer Robert D. , Cockett Abraham T. K.
Published: October 2006

The unprecedented ability of cyclosporin A, when given for six days at a dose of 25 mg/kg/d or 50 mg/kg/d, to cause a marked and sustained increase in renal glutathione (GSH) concentration in rat kidney is described. This response was particular to the kidney insofar as the GSH concentration in the liver was not increased in response to a lower dose of cyclosporin and was decreased in the liver of animals treated with the higher dose of the drug. The increase in kidney GSH concentration did not appear to be due to an increased rate of production or to an inhibition of the degradation of the tripeptide. This suggestion is based on the finding that the activities of the GSH synthesis pathways, GSSG‐reductase and γ‐glutamylcysteine synthetase, were unchanged or decreased, respectively, and those of the catabolic enzymes, GSH‐peroxidase and γ‐glutamyltranspeptidase, were unchanged or increased, respectively.

It is suggested that the elevation of renal GSH content in the face of diminished synthetic capacity and an apparent increased utilization may result from an enhanced uptake of GSH as the result of alterations caused by cyclosporin in the renal transport system.
Toluene‐induced alterations in rat synaptosomal membrane composition and function
Authors: Le Bel Carl P. , Schatz Robert A.
Published: October 2006

Toluene is a widely used organic solvent that can produce acute central nervous system (CNS) effects. Since toluene reaches relatively high concentrations in the CNS and is extremely lipophilic, we investigated its effects on rat brain membrane composition and function. Toluene (1 g/kg, lh) did not alter total brain microsomal phospholipid (PL) or cholesterol (CL) content. However, synaptosomal PL was decreased (24%), while synaptosomal CL was unaltered. The PL/CL ratio, an indirect index of membrane fluidity, did not change, suggesting that toluene did not affect membrane fluidity. Fluorescence polarization studies employing 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) showed that toluene did not alter synaptosomal membrane fluidity after administration in vivo (1 g/kg) or in vitro (0.5 to 5.0 mM). Dose‐response and time‐course studies showed that toluene maximally decreased synaptosomal PL after 1 g/kg, 1 h. The dose‐response and time‐course studies also showed that the toluene‐induced decreases in PL were a result of specific decreases in phosphatidylethanolamine (PE). Since PE was decreased, we assessed whether toluene altered synaptosomal membrane function by investigating phospholipid methylation, a reaction which uses PE as its initial substrate. Toluene decreased the incorporation of methyl groups into lipid when [3H]‐methionine was used as the methyl donor, but did not affect methylation when [3H]‐adenosylmethionine was the methyl donor. These data suggest that toluene‐induced specific decreases in synaptosomal PE and inhibition of phospholipid methylation may alter normal synaptic function and play a critical role in the mechanism(s) of action of toluene's CNS effects.
Sensitivity of selected drug biotransformation enzymes to dietary protein levels in adult F344 male rats
Authors: Butler L. E. , Dauterman W. C.
Published: October 2006

Adult male Fischer 344 rats were fed inadequate, adequate, and excessive quantities of dietary protein (8, 12, and 22%, respectively) for a period of 14 days. An increase in dietary protein did not increase liver weight but resulted in an increase in cytochrome P‐450 content and 7‐ethoxycoumarin O‐deethylase activity. No significant difference in glutathione S‐transferase activity was observed at the three protein levels. The in vivo hepatotoxicity of bromobenzene increased with an elevation in dietary protein intake from 12 to 22%. These data from mature rats follow trends similar to findings we have reported previously with juvenile rats.
TCDD (2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin) is a tight binding inhibitor of cytochrome p‐450D
Authors: Voorman Richard , Aust Steven D.
Published: October 2006

Based on data indicating that compounds which induce cytochrome P‐450d also bind to the enzyme (R. Voorman and S. D. Aust (1987). Toxicol. Appl. Pharmacol. 90, [69–78], we investigated the inhibition of cytochrome P‐450d‐dependent estradiol 2‐hydroxylase by (2,3,7,8‐tetrachlorodibenzo‐p‐dioxin TCDD), using ligand‐free cytochrome P‐450d from isosafrole‐treated rats. Since maximum inhibition of estradiol 2‐hydroxylase occurred at TCDD concentrations comparable to the concentration of enzyme (50 nM), a modified form of steady‐state kinetic analysis was used. Using I50 =Et/2 + Ki where Et =total enzyme concentration), we showed that TCDD inhibited cytochrome P‐450d‐dependent estradiol 2‐hydroxylase activity with Ki equal to 8 nM. Association of TCDD with P‐450d occurred within 2 min of inhibitor addition. Therefore, TCDD can be considered a tight binding inhibitor of cytochrome P‐450d.
Effects of hyperthermia on xanthine oxidase activity and glutathione levels in the perfused rat liver
Authors: Skibba Joseph L. , Stadnicka Anna , Kalbfleisch John H. , Powers Robert H.
Published: October 2006

The hepatotoxic effects of hyperthermic liver perfusion were investigated in male Fischer 344 rat livers. Perfusions were carried out at 37, 41, 42, 42.5, and 43°C for 2 hr. During the 2 hr, the perfusate was analyzed for activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), N‐acetyl‐β‐glucosaminidase (NAG), and glutathione (GSH), oxidized glutathione (GSSG), allantoin, and potassium. After perfusion, each liver was homogenized and analyzed for total xanthine oxidase (XO) activity, percentage type‐D and type‐O XO, and total GSH content. Perfusate AST, LDH, NAG, and potassium levels were increased significantly with time and were significantly different in all hyperthermic perfusions from the 37°C perfusion values by the end of the perfusion. Perfusate GSH + GSSG levels were increased significantly in all hyperthermic perfusions after 60 min. Liver GSH levels were significantly lowered following perfusion at hyperthermic temperatures. There was a temperature‐dependent increase in the percentage of XO in the type‐O form following perfusion at hyperthermic temperatures, which was strongly and positively correlated with the loss of hepatic GSH. These data support the hypothesis that hyperthermic toxicity to the liver is the result of oxidative stress brought about by conversion of XO to the type‐O form.
Carbon tetrachloride metabolism in partially hepatectomized and sham‐operated rats pre‐exposed to chlordecone (kepone)
Authors: Young Robert A. , Mehendale Harihara M.
Published: October 2006

The potentiation of CCl4 toxicity by pre‐exposure to chlordecone (CD) is well established. Chlordecone‐induced metabolism of CCl4 and suppressed hepatocellular repair have been offered as possible mechanisms for this potentiation. Recent work using the partially hepatectomized (PH) rat as a model for an actively regenerating liver has provided supportive evidence for the latter hypothesis. The present study was initiated to determine if metabolism and disposition of 14CCl4 is altered in the PH rat, and if this is a contributing factor to the reported protective effect afforded by the PH procedure. Male Sprague‐Dawley rats (150–175 g) maintained on dietary CD (10 ppm) for 15 days were partially hepatectomized or sham‐operated (SH) on day 15. Another group of CD‐pretreated rats received 0.9% CoCl2 (60 mg/kg, sc, qd for 2 days) in lieu of the surgical procedure. On day 16 the rats were challenged with a single dose of CCl4 (100 μL/kg, ip) containing 20 μCi 14CCl4. A radiolabel inventory consisting of exhaled 14CCl4, 14CO2 production, total hepatic 14C, free 14CCl4 and covalently bound 14C was taken over a 6‐hr time period. Lipid peroxidation and serum enzyme activities [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] were measured as indices of toxicity. Neither CD pretreatment alone nor CoCl2 treatment alone produced significant alterations in metabolism of low dose (100 μl/kg) CCl4. No significant difference in 14CCl4 recovery or 14CO2 production was detected for PH versus SH rats. Hepatic 14CCl4‐derived 14C (per gram tissue) was greater in PH rats. Values for free 14CCl4, covalently bound 14C, and lipid peroxidation were similar for SH and PH rats. The data suggest that metabolism and disposition of CCl4 are not altered by PH. Thus the PH rat is a valid model for studying the CD + CCl4 interaction in an actively regenerating liver, and the protection afforded by partial hepatectomy is not due to a reduction in hepatic uptake or bioactivation of CCl4.
Feprazone: An inducer of the P450 II B family of proteins in the rat
Authors: Smith Jonathan N. , Ayrton Andrew D. , Chown Janice , Lewis David F. V. , Ioannides Costas
Published: October 2006

The ability of feprazone to induce the hepatic microsomal mixed‐function oxidases was investigated in the rat, with emphasis being placed on the nature of the cytochrome P‐450 family induced. Treatment with feprazone enhanced the p‐hydroxylation of aniline and the dealkylations of benzphetamine and pentoxyresorufin but had no effect on the O‐deethylation of ethoxyresorufin. The same treatment had no major effect on total cytochrome P‐450 levels but increased the spectral interaction of metyra‐pone with reduced cytochrome P‐450. Immunoblots employing monospecific polyclonal antibodies revealed that feprazone induces the apoprotein levels of the P450 II B, but not of the P450 I, family. It is concluded that feprazone is an inducer of the rat hepatic mixed‐function oxidase system showing selectivity toward the P450 II B family.
Exposure of Pulmonary Artery Endothelial Cells to Nitrogen Dioxide Activates Phospholipase A1
Authors: Bhat G. B. , Patel J. M. , Block E. R.
Published: October 2006

Phospholipase A1, A2, and C and diacylglycerol lipase activities were measured in cell sonicates after exposing confluent monolayers of porcine pulmonary artery endothelial cells to 5 ppm NO2, a toxic constituent of environmental pollution, for 24 and 48 hr. There was a significant increase (2.25‐fold) in phospholipase A1 activity in 24 and 48 hr NO2‐exposed cells, whereas activities of phospholipases A2 and C and diacylglycerol lipase were comparable to control cells at both time points. When endothelial cells were prelabeled with [3H]‐arachidonic acid and then exposed to NO2 for 48 hr, increased counts were recovered from cell lysophospholipids with concomitant decreased recovery of counts from cell phosphatidylcholine and phosphatidylethanolamine. These results demonstrate that NO2 exposure results in specific activation of phospholipase A1.
Metabolism and toxicity of xenobiotics in the adrenal cortex, with particular reference to 7,12‐dimethylbenz(a) anthracene
Authors: Hallberg Einar
Published: October 2006

The adrenal cortex contains high amounts of detoxifying enzymes, as well as generators and protectors of reactive oxygen species. The high content of cytochrome P‐450 enzymes in the adrenal cortex together with its remarkable tendency to accumulate hydrophobic substances probably contributes to the extraordinary vulnerability of the gland to a number of xenobiotics. The best studied adreno‐corticolytic compounds are the potent carcinogen 7, 12‐dimethylbenz(a)anthracene (DMBA) and its liver metabolite 7‐hydroxymethyl‐12‐methylbenz (a)anthracene (7‐OHM‐12‐MBA). Adrenocorticolysis generated by these agents in vivo as well as in vitro demonstrates high regioselective requirements and is strongly influenced by the presence of ACTH, steroids, cytochrome P‐450 inhibitors and antioxi‐dants. Furthermore, 7‐OHM‐12‐MBA has been demonstrated to uniquely generate selective and massive oxidation of mitochondrial glutathione in cultured rat adrenal cells.

The DMBA‐induced adrenocorticolysis is thoroughly discussed in this review with particular emphasis on the metabolism of DMBA and the influence of various effectors. A working hypothesis involving a possible peroxidative mechanism is also presented.
The effects of haloalkene cysteine conjugates on cytosolic free calcium levels in suspensions of rat renal proximal tubules
Authors: Groves Carlotta E. , Lock Edward A. , Schnellmann Rick G.
Published: October 2006

Disturbances in intracellular calcium homeostasis may play a role in the injury induced by various haloalkene cysteine conjugates. The effects of S‐(1,2,3,4,4‐pentachloro‐1,3‐butadienyl)‐L‐cysteine (PCBC), S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC), and S‐(1,1,2,2‐tetrafluoroethyl)‐L‐cysteine (TFEC) on cytosolic free calcium levels were examined in suspensions of rat renal proximal tubules. Cytosolic free calcium levels, measured with fura 2, in control tubules, were 112 ± 3 nM and increased more than 200% within 1 minute after exposure to the calcium ionophore ionomycin (0.005 mM). PCBC (0.1 mM) increased cytosolic free calcium levels 18% after 5 minutes, while tubular oxygen consumption was unaffected. DCVC (1 mM) did not alter tubular cytosolic free calcium levels or oxygen consumption under similar conditions. TFEC (1 mM) increased cytosolic free calcium levels 36%, had no effect on basal oxygen consumption, and decreased nystatin‐stimulated oxygen consumption 30% after 5 minutes. TFEC increased cytosolic free calcium levels in tubules incubated in a nominally calcium‐free buffer but not in a calcium containing buffer in the presence of EGTA. The data suggest that the TFEC‐induced increase in cytosolic free calcium levels may result from an influx of extracellular calcium or from inhibition of calcium efflux. The increase in cytosolic free calcium levels preceded changes in basal oxygen consumption in tubules exposed to PCBC and TFEC. This study shows that an increase in cytosolic free calcium levels is an early event following PCBC and TFEC but not DCVC exposure.
Diethylstilbestrol potentiates and testosterone antagonizes the action of 3‐methylcholanthrene on benzo(a) pyrene metabolism in hep G2 cells
Authors: Moore Susan Mcafee , Lamartiniere Coral A.
Published: October 2006

We have used the human hepatoma cell line, Hep G2, to examine the ability of hormones and xenobiotics to modulate the hepatic induction of benzo(a)pyrene hydroxylase and epoxide hydrolase. Hep G2 cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum. 3‐Methylcholanthrene, diethylstilbestrol, testosterone propionate, and combinations of 3‐meth‐ylcholanthrene, and each of the hormones were added directly to the culture media. We subsequently studied the metabolism of benzo(a)pyrene using cell lysates of the Hep G2 cells. Metabolites were quantitated by high‐performance liquid chromatography (HPLC) using fluorodetection. Exposure to 3‐methyl‐cholanthrene alone resulted in an eightfold increase in total benzo(a)pyrene metabolites with a change of the predominant metabolite from the 3‐hydroxy‐benzo(a)pyrene to the carcinogenic pathway of the benzo(a)pyrene‐7,8‐diol. Diethylstilbestrol and testosterone propionate resulted in small, but significant, decreases in metabolism of benzo(a)pyrene. When exposed in combination with 3‐methyl‐cholanthrene, testosterone propionate antagonized and diethylstilbestrol potentiated the metabolism of benzo(a)pyrene. 3‐Methylcholanthrene, diethylstilbestrol, and combinations of 3‐methylcholanthrene and diethylstilbestrol or testosterone propionate resulted in increased epoxide hydrolase activity as compared to controls. These results, carried out in a human hepatoma cell line, lend support to a concern for potentiated toxicity and carcinogenicity following exposure to complex chemical mixtures.
Spectroscopic quantitation of cytochrome P‐450 in human lung microsomes
Authors: Wheeler Clyde W. , Guenthner Thomas M.
Published: October 2006

The cytochrome P‐450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide‐complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P‐450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.
Role of lipid metabolism in cell killing by calcium plus ionophore A23187
Authors: Shier W. Thomas , Dubourdieu Daniel J. , Wang Huei‐Hsiang L.
Published: October 2006

Cultured fibroblasts treated with divalent cation ionophore A23187 in the presence of extracellular calcium provide a useful model system for studying mechanisms of cell death associated with elevated intracellular calcium concentrations. Cell death induced by A23187 plus calcium can be conveniently monitored as membrane permeabilization to Trypan blue dye. Because lipids are a major component of cell membranes and play an important role in determining membrane permeability, the present study was initiated to identify changes in cell lipid composition that occur during membrane permeabilization induced by calcium plus A23187. The percent label in each of the major structural lipids in biosynthetically labeled NIH3T3 fibroblasts changed < 10% during the time course of membrane permeabilization. During the course of membrane permeabilization there was significantly increased label in lysophosphatidylinositol and lysophosphatidylcholine and reduced label in phosphatidylinositol 4,5‐bisphosphate. The time course of these changes corresponded to that of the arachidonic acid release response stimulated by calcium plus A23187, not to the time course of membrane permeabilization, which occurs later. These observations are consistent with lipid metabolism induced by A23187 plus calcium playing only a possible regulatory or intermediatory role in membrane permeabilization, rather than causing direct permeabilization of the lipid phase of the membrane.
Characterization of covalent binding of [14C]‐2‐chloro‐4‐acetotoluidide to microsomes of starling liver and kidney
Authors: Giri Shri N. , Siegel David M.
Published: October 2006

In this study, we have characterized the covalent binding of [14C]‐2‐chloro‐4‐acetotoluidide (CAT) radioactivity to microsomes of starling liver and kidney. The maximal velocity (Vmax) of covalent binding and apparent Michaelis constant (Km) for both tissues were similar. The Vmax for liver and kidney were 52.8 and 68.9 pmol/min/mg protein, and the apparent Kms were 0.54 and 0.87 mM, respectively. The covalent binding of radioactivity to heat‐denatured microsomes of liver and kidney was reduced by 62% and 15%, respectively. Incubation at 0°C reduced the binding by 80% to liver and 70% to kidney microsomes. Absence of nicotinamide adenine dinucleotide phosphate (NADP) and molecular O2 reduced the binding to liver microsomes by 36 and 53%, as opposed to 28% increase and 26% decrease in binding to kidney microsomes, respectively. Inducers of cytochrome P450 monooxygenase (P450), phenobarbital, and 3‐methylcholanthrene (3‐MC), had opposite effects on the covalent binding of [14C]‐CAT radioactivity to hepatic and renal microsomes. Phenobarbital increased the binding to hepatic microsomes by 100% and had no effect on binding to renal microsomes. 3‐MC, on the other hand, increased the binding to kidney microsomes by threefold and had no effect on the binding to hepatic microsomes. SKF 525A, an inhibitor of P450, inhibited the binding to hepatic microsomes by 60% at 0.5 mM but failed to have any effect on binding to renal microsomes. α‐Naphthoflavone, another inhibitor of P450, had no effect on the covalent binding of [14C]‐CAT radioactivity to microsomes of either tissue. Addition of cysteine and glutathione in the incubation mixture inhibited the covalent binding of radioactivity to both liver and kidney microsomes in a concentration‐dependent manner. It was concluded that hepatic and renal microsomes of starling behave differently with respect to the effects of P450 inducers and inhibitors, heat denaturation, NADPH dependence, and presence of molecular oxygen on bioactivation of [14C]‐CAT and its subsequent covalent binding to microsomal proteins.
In vitro inhibition of rat platelet aggregation by ochratoxin A
Authors: Omar Rabeea F. , Randell Edward , Rahimtula Anver D.
Published: October 2006

The mycotoxin ochratoxin A (OA) consists of 5‐chloro‐3‐methyl‐3,4‐dihydro‐8‐hydroxyisocoumarin moiety linked by an amide bond to β‐L‐phenylalanine. When added to washed rat platelets in vitro, OA caused a dose‐dependent inhibition of aggregation induced by agonists such as adenosine diphosphate (ADP) or thrombin. The aggregatory response induced by prior addition of an agonist was also reversed in a dose‐dependent manner by OA. Inhibition of aggregation appeared to be irreversible since exposure of platelets to OA followed by several washings removed most of the mycotoxin associated with the platelets but did not diminish the inhibitory response. Serotonin secretion from dense granules and arachidonic acid release from membrane phospholipid (especially phosphatidylcholine) as well as its further metabolism were also inhibited by OA. These results suggest that a disruption of the platelet plasma membrane structure by OA is probably responsible for inhibition of the primary and secondary phases of aggregation.
Lack of desensitization of muscarinic receptor–mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase
Authors: Abdallah Elsayed A. M. , El‐Fakahany Esam E.
Published: October 2006

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor downregulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP‐induced muscarinic receptor down‐regulation and receptor desensitization.
Intracellular calcium chelators and oxidant‐induced renal proximal tubule cell death
Authors: Schnellmann Rick G.
Published: October 2006

The effect of intracellular calcium chelators on rabbit renal proximal tubule (RPT) cell death induced by t‐butyl hydroperoxide (TBHP) and H2O2 was examined. Preincubation of RPT suspensions with 50 μM QUIN 2/AM completely prevented TBHP (0.5 mM) and H2O2 (2 mM) induced cell death [i.e., release of lactate dehydrogenase (LDH)]. QUIN 2/AM, BAPTA/AM, EGTA/AM, and FURA 2/AM, at 5 μM, decreased LDH release (at 6 hr) from 41% to 4%, 21%, 26%, and 33%, and decreased lipid peroxidation (at 1 hr) from 1.0 to 0.1, 0.4, 0.6, and 0.8 nmol MDA/mg protein, respectively, after TBHP exposure. Since oxidant‐induced lipid peroxidation and cell death are iron‐dependent in this model, these results suggest that the intracellular calcium chelators inhibit cell death by chelating iron.
Modulation of transcription in rat liver nuclei in vitro by a diol epoxide of benzo[a]pyrene
Authors: Shah G. M. , Bhattacharya R. K.
Published: October 2006

Treatment of isolated rat liver nuclei with 7β, 8α‐dihydroxy‐9α, 10α‐epoxy‐7, 8, 9, 10‐tetrahy‐drobenzo[a]pyrene, the ultimate carcinogenic metabolite of benzo[a]pyrene, resulted in inhibition of transcription as measured by radioactive precursor incorporation into RNA. The mechanism of inhibition as analyzed by use of different types of inhibitors suggested that the carcinogen acted on both the major components of transcription machinery, that is, the template chromatin and the enzyme RNA polymerases. This action correlates well with the observations made after administration of benzo[a]pyrene to rats.
Effect of N‐methyl‐N′‐Nitro‐N‐nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach
Authors: Bagewadikar R. S. , Bhattacharya R. K.
Published: October 2006

Administration of the carcinogenic N‐nitroso compound, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in drinking water (0.5 mg/mL) to male Wistar rats for 1 week caused impairment of in vivo and in vitro incorporation of [14C]leucine into stomach mucosal protein. This impairment gradually returned to normal after 4 weeks. Uptake of [14C]leucine into mucosal protein was significantly inhibited after in vitro treatment of stomach mucosa with the carcinogen. Addition of the N‐nitroso compound in a cell‐free system using postmitochondrial supernatant prepared from stomach mucosa also showed inhibition of amino acid incorporation. Using a more defined system consisting of purified polyribosome from stomach mucosa and pH 5 enzyme fraction derived from liver it was further demonstrated that the carcinogen purturbed protein synthesizing ability of polyribosome, under both in vivo and in vitro treatment conditions. In these respects this carcinogen has similar action on the target tissue of stomach as in the liver, although the in vivo effect may be related more to toxicity than carcinogenicity.
Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1
Authors: Gemzik Brian , Greenway Denise , Nevins Cheryl , Parkinson Andrew
Published: October 2006

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51‐kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone‐16α‐carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51‐kDa protein. Treatment of male rats with Aroclor 1254 induced the 51‐kDa protein, but suppressed the 50‐kDa form. In addition to their changes in response to inducers, the 50‐ and 51‐kDa proteins also differed in their developmental expression. For example, the 50‐kDa protein was not expressed until weaning (3 weeks), whereas the 51‐kDa protein was expressed even in 1‐week‐old rats. At puberty (between weeks 5 and 6), the levels of the 50‐kDa and 51‐kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male > female) in the levels of both proteins. Changes in the level of the 51‐kDa protein were paralleled by changes in the rate of testosterone 2β, 6β‐, and 15β‐hydroxylation. In male rats, the marked increase in the levels of the 50‐kDa protein between weeks 2 and 3 coincided with a three‐ to four fold increase in the rate of testosterone 2β‐, 6β‐, and 15β‐hydroxylation, which suggests that the 50‐kDa protein catalyzes the same pathways of testosterone oxidation as the 51‐kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51‐kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51‐kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2β‐, 6β‐, and 15β‐hydroxylation of testosterone.
Biological action of lantadene C, a new hepatotoxicant from Lantana camara var. aculeata
Authors: Sharma Om P. , Vaid Jagdish , Pattabhi Vasantha , Bhutani Kamlesh K.
Published: October 2006

Lantadene C (22β‐2‐methylbutanoyloxy ‐3‐oxoolean‐12‐en‐28‐oic acid) isolated from the leaves of the hepatotoxic plant Lantana camara var. aculeata (Red) has been found to be identical with dihydrolan‐tadene A reported earlier. Molecular structure of lantadene C has been deduced from single crystal X‐ray diffraction analysis. It resembles lantadene A in the pentacyclic portion of the molecule but differs in the side chain region. Atom C‐34 is cis to C‐35 in lantadene C but is transin lantadene A. Semisynthetic lantadene C was prepared by catalytic hydrogenation of lantadene A. Lantadene C was obtained in two forms, I and II. Form I was crystalline while form II was amorphous. Unlike lantadene A, both form I and II of lantadene C elicited strong hepatotoxic response in guinea pigs associated with decrease in fecal output, feed intake, hepatomegaly, hepatic injury at the cellular and subcellular level, increase in plasma bilirubin, and acid phosphatase activity. All the clinical signs, hepatic lesions, and changes in blood plasma typified lantana toxicity. This is the first report on the hepatotoxicity of lantadene C. The interrelation of molecular structure and biological activity of lantadene A and C has been discussed.
The flavin‐containing monooxygenase in mouse lung: Evidence for expression of multiple forms
Authors: Venkatesh Krishnappa , Blake Bonnie , Levi Patricia E. , Hodgson Ernest
Published: October 2006

The flavin‐containing monooxygenase (FMO) was purified from mouse lung microsomes. On SDS‐PAGE, the purified enzyme separated as two bands, a major band of 58,000 daltons and a minor band of 59,000 daltons. Antibodies to mouse liver FMO cross‐reacted with both bands in the purified preparations, whereas antibodies to rabbit lung FMO cross‐reacted only with the major band. In microsomal preparations the major band was recognized by both antibodies, but neither antibody detected the minor band in microsomes. A cDNA encoding the pig liver FMO hybridized with mRNA isolated from mouse liver, kidney, and lung, whereas cDNA encoding the rabbit lung FMO hybridized only with mouse lung and kidney mRNA. Thermal stability studies showed that the FMO preparation purified from mouse lung consisted of a heat‐stable and a heat‐labile component. The heat‐labile component of lung FMO was inhibited competitively by imipramine, whereas the heat‐stable component was insensitive to the presence of imipramine. Immunoprecipitation of purified mouse lung FMO with anti‐rabbit lung FMO completely removed the protein band reactive to anti‐rabbit lung FMO while leaving reactivity to anti‐liver FMO. The catalytic and immunochemical differences seen between FMO from rabbit lung and mouse lung appear to result from the expression of at least two forms of FMO in the mouse lung, one similar to the rabbit pulmonary form and one similar to the major mouse liver form of FMO.
A chaperone‐mimetic effect of serum albumin on rhodanese
Authors: Jarabak Rebecca , Westley John , Dungan Joseph M. , Horowitz Paul
Published: October 2006

Reactivation of denatured rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was found to be aided by the presence of serum albumin. Both the rate and the extent of reactivation of the urea‐denatured enzyme were optimal at low rhodanese and moderate serum albumin concentrations. Similarly, stabilization of the sulfurtransferase activity of rhodanese that had been partially unfolded at 40°C was aided by the presence of serum albumin. All the observations are in accord with a model in which enzyme that has been partially refolded from the urea‐denatured state or partially unfolded thermally interacts directly with serum albumin in a way that prevents rhodanese self‐association. Serum albumin thus acts as a molecular chaperone in these systems.
2,3,7,8‐tetrachlorodibenzo‐P‐dioxin induced alterations in protein phosphorylation in guinea pig adipose tissue
Authors: Enan Essam , Matsumura Fumio
Published: October 2006

An in situ (explant tissue culture) model has been developed to study the effect of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), hormones, and growth factors either alone or in combination. In our model system, the effect of TCDD on protein phosphorylation was greatly affected by the presence or the absence of externally added D‐glucose. In the presence of a physiologically relevant level of glucose (13.3 mM), TCDD clearly stimulated protein phosphorylation as in the case of in vivo data. However, in the absence of Dglucose TCDD clearly inhibited protein phosphorylation. On the other hand, TCDD reduced the glucose uptake activity in isolated adipose tissue either in the presence or absence of D‐glucose (13.3 mM). Therefore, the TCDD‐induced reduction of glucose transport does not appear to be related directly to the simultaneous rise in protein phosphorylation. For comparison, several agents which are known to affect protein phosphorylation were tested. These hormonal agents generally affected the TCDD‐untreated adipose tissues in the manner expected from their known actions, indicating that this in situ model is an adequate system to study their independent actions. The TCDD‐treated adipose tissue samples showed only mild or insignificant response to these hormonal stimuli. In terms of the changes in the pattern of protein phosphorylation activities, the action of TCDD appeared to resemble that of EGF and T3. Since under in situ conditions no agents such as EGF or T3 can be expected to be present, the observed TCDD‐induced changes are suggestive of the basic intracellular changes in cellular activities. The types of TCDD‐induced protein kinases appear to be protein tyrosine kinases and protein kinase C.
Effect of anti‐inflammatory agents on ricin‐induced macrophage toxicity
Authors: Naseem Syed M. , Pace Judith G.
Published: October 2006

The toxicity of ricin in susceptible cells is well characterized biochemically, but the pathophysiological implications of its toxicity and the immune response to ricin challenge in the lung are unknown. Incubating macrophage cell line with ricin (1 pM‐10 nM) for 4 hours markedly inhibited 3H‐leucine incorporation (acid insoluble) into protein (>95%, at 1 nM) without affecting the acid‐soluble radioactivity. In spite of increased uptake of total thymidine (141×13.5%) and total uridine (135×17.2%), DNA synthesis in ricin‐treated cells was progressively inhibited although RNA synthesis was not affected. Fluocinolone (an anti‐inflammatory glucocorticoid) pretreatment increased the ricin‐induced inhibition of protein synthesis. The synergistic effect of fluocinolone on ricin‐induced protein synthesis inhibition was due to an increased binding (167%, p < 0.01) and internalization (134×12%, p < 0.025) of ricin. Partial protection from ricin‐induced inhibition of protein synthesis by indomethacin (nonsteroidal, anti‐inflammatory agent) was due to decreased binding and internalization of ricin. These results show that macrophages are sensitive to ricin and that pharmacologically active drugs may regulate ricin's toxicity, perhaps by controlling synthesis and release of certain mediators of fast death.
Purification and properties of a diisopropyl‐fluorophosphatase from squid Todarodes pacificus steenstrup
Authors: Wang Fei , Xiao Meizhen , Mu Shaofeng
Published: October 2006

A diisopropyl‐fluorophosphatase (DFPase) was purified from brain and ganglia of squid Todarodes pacificus steenstrup. The DFPase had a preference in hydrolysis toward diisopropylphosphorofluoridate (DFP). It also was able to hydrolyze O‐1,2,2‐trimethylpropyl methylphosphofluoridate (soman) and O‐isopropyl methylphosphonofluoridate (sarin) at nearly equal hydrolytic rates but only 1/10 that of DFP. The hydrolytic activity toward diethyl‐p‐nitrophenylphosphate (paraoxon) was very low compared with DFP, so man, and sarin. The DFPase was purified 330‐fold to a specific activity of 18,300 n mol/min/mg protein. Its molecular weight was 34,000 dalton determined by gel‐filtration chromatography. Mn2+ stimulation of the DFPase was not observed when DFP and soman were the substrates, but with sarin, the rate increased onefold in the presence of 1.0 mM of Mn2+. Ethylenediamine tetraacetic acid disodium (EDTA‐Na2) at 0.05 M inhibited the DFPase activity about 30%. It could be concluded that this DFPase belongs to the squid‐type DFPase.
Differential effects of short‐term lindane administration on parameters related to oxidative stress in rat liver and erythrocytes
Authors: Bainy A. C. D. , Arisi A. C. M. , Azzalis L. A. , Simizu K. , Barros S. B. M. , Videla L. A. , Junqueira V. B. C.
Published: October 2006

Parameters related to oxidative stress in rat liver and erythrocytes were studied after short‐term administration (60 and 90 days) of 1000 ppm of lindane in the diet. Lindane induced an oxidative stress condition in the liver, which is related to an enhancement in microsomal NADPH‐cyto‐chrome c reductase and NADPH oxidase activities, superoxide radical formation and cytochrome P450 content, produced independently of the time of treatment. Also, decreased activities of glutathione peroxidase and catalase were concomitantly observed. Although these changes were paralleled by an increase in lipid peroxidation indices, such as production of thiobarbituric acid reactants and spontaneous chemiluminescence, no evidence of liver injury was obtained. Lindane treatment did not exert quantitatively important changes in the pro‐oxidant/anti‐oxidant status of the erythrocyte, with reduction in the red blood cell mass possibly reflecting actions of the insecticide on the eryth‐ropoietic process.