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Biomedical subjects

H G Shertzer

Publications and source records attributed to H G Shertzer.

16 recordsLinked to original sources
PubMed1992

Evaluation of iron binding and peroxide-mediated toxicity in rat hepatocytes.

A novel assay was developed to determine subnanomolar amounts of Fenton-reactive iron (FRI) in biological tissues. FRI represents that pool of iron that is redox active and capable of participating in a model Fenton reaction. The FRI was used to identify a kinetically-defined cellular iron binding site. This site displays positive cooperativity, with apparent kinetic constants of Kd = 10.6 microM, Bmax = 20.7 nmol/mg protein, and the Hill coefficient = 1.4. After addition of exogenous ferrous ammonium sulfate to hepatocytes, binding occurred within a few seconds and was stable for at least an hour. Free extracellular iron, but not bound iron, stimulated lipid peroxidation in hepatocytes. In contrast, bound but not free iron produced a concentration-dependent increase in tert-butyl hydroperoxide (TBH)-mediated toxicity, suggesting the toxicological relevance of bound, rather than free iron. Furthermore, the hydroxyl radical scavengers mannitol and 2-deoxyribose inhibited Fe2/TBH-mediated lipid peroxidation, but not cell killing, suggesting that hydroxyl radical may not be involved in the critical toxic event. The divalent cations Mn2+ and Co2+ inhibited iron-mediated hepatocyte killing in the presence of TBH, but only if added prior to Fe2+. Mn2+, but not Co2+, inhibited Fe(2+)-mediated lipid peroxidation regardless of the order of addition. These results indicate the existence of a specific, kinetically-defined cellular iron binding site. Such binding is involved in peroxide-mediated toxicity, but independent of lipid peroxidation. The specific nature of this site and involvement with other forms of chemical intoxication or cellular iron homeostasis are unknown.

Animals↗
PubMed1992

Menadione-mediated membrane fluidity alterations and oxidative damage in rat hepatocytes.

Menadione toxicity in isolated rat hepatocytes was mitigated by the antioxidant 4b,5,9b,10-tetrahydroindeno[1,2-b]indole at low concentrations (less than 100 microM), but not at high concentrations (greater than 200 microM) of menadione. When hepatocytes were incubated with menadione, there was a time-dependent and concentration-dependent inhibition of lipid peroxidation in intact cells, as well as an increase in the antioxidative potency of acetone extracts, suggesting that metabolites of menadione could inhibit oxidative stress, and that at high menadione concentrations a different mechanism was involved in cytotoxicity. A possible mechanism was suggested by the ability of acetone extracts from hepatocytes that had been incubated with menadione to increase osmotic fragility in red blood cells. This increase correlated with an increase in membrane fluidity in red blood cells, determined by flourescence polarization using the membrane probe 1,6-diphenyl-1,3,5-hexatriene. At 200 microM menadione, an increase in membrane fluidity was also observed in hepatocytes. The thiol dithiothreitol protected hepatocytes from 50 microM menadione toxicity, but not from greater than or equal to 100 microM menadione. The results suggest that while oxidative stress and arylation may be the critical mechanisms of toxicity at low menadione concentrations, at higher concentrations another mechanism such as enhanced membrane fluidity is operative.

Animals↗
PubMed1992

"Oxidative stress" response in liver of an untreated newborn mouse having a 1.2-centimorgan deletion on chromosome 7.

The c14CoS/c14CoS mouse has a homozygous deletion of about 1.2 cM on chromosome 7 that includes the albino (c) locus. The untreated 14CoS/14CoS newborn has been reported to exhibit a marked transcriptional activation of the hepatic NAD(P)H:menadione oxidoreductase (Nmo-1; DT diaphorase; quinone reductase; azo dye reductase) gene, as well as elevated UDP glucuronosyl-transferase (UGT1*06) and glutathione transferase (GT1) activities, when compared with the cch/cch wild-type and the cch/c14CoS heterozygote. We show here that the newborn hepatic activities of seven enzymes that play a role in the oxidative stress response--NMO1, UGT1*06, GT1, copper-zinc superoxide dismutase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase--are increased 1.5- to 25-fold in 14CoS/14CoS, as compared with ch/ch and ch/14CoS mice. The activities of four additional enzymes having no known association with the oxidative stress response--benzo[a]pyrene hydroxylase (CYP1A1, cytochrome P(1)450), acetanilide 4-hydroxylase (CYP1A2, cytochrome P(3)450), lactate dehydrogenase (LDH), and NADPH-cytochrome c reductase--are not significantly different among the three genotypes. These data suggest that there exists an "oxidative stress" response in the untreated 14CoS/14CoS newborn. We postulate that a chromosome 7 regulatory gene, which we have named Nmo-1n, might encode a trans-acting negative effector of the Nmo-1 gene, and genes corresponding to the other elevated enzymic activities described above. When both copies of Nmo-1n are deleted, as is the case in 14CoS/14CoS mice, a battery of genes involved in oxidative stress is released from negative control and becomes activated--despite the absence of any apparent oxidative insult by foreign chemicals.

Animals↗
PubMed1991

Relationship of membrane fluidity, chemoprotection, and the intrinsic toxicity of butylated hydroxytoluene.

In isolated rat hepatocytes, many chemicals elicit toxicity which is inhibitable by antioxidants such as butylated hydroxytoluene (BHT). Although BHT protection is evident at concentrations of less than about 50 nmol/mg protein, higher concentrations exhibit intrinsic concentration-dependent toxicity, which involves mitochondrial dysfunction. We evaluated the possibility that both chemoprotection and intrinsic toxicity could be explained by a common mechanism involving alterations in the physical properties of cellular membranes. In the red blood cell (RBC) osmotic fragility assay, BHT at less than 60 nmol/mg protein protected against osmotic fragility; however, BHT at higher concentrations enhanced osmotic fragility such that total osmolysis occurred at 135 nmol/mg. The BHT-mediated alterations in osmotic fragility correlated with changes in membrane fluidity, determined by fluorescence polarization of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Protection from osmolysis correlated with decreased fluidity, while enhanced RBC fragility correlated with increased fluidity. In rat hepatocyte suspensions, high BHT concentrations also permeabilized the plasma and mitochondrial membranes to enzyme leakage, and these effects were accompanied by enhanced membrane fluidity. Although other mechanisms may be operative, alterations in membrane fluidity appear to be, in part, responsible for the observed chemoprotective effects at low concentrations, and intrinsic toxicity at higher concentrations of BHT.

Animals↗
PubMed1991

Mechanisms of chemical mediated cytotoxicity and chemoprotection in isolated rat hepatocytes.

Although N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methylmethanesulfonate (MMS) cause injury and malondialdehyde formation in rat hepatocytes, MNNG toxicity is much more sensitive to inhibition by antioxidants. In order to quantify the relationship between toxicity and antioxidation potential, we compared 14 antioxidants that protected against MNNG and MMS toxicity. Chemoprotection was quantified as the concentration that delayed by 1 h the decline in trypan blue exclusion to less than or equal to 50%. While chemoprotection against MNNG and antioxidant efficacy were directly related (R = 0.86), chemoprotection against MMS and antioxidant efficacy were unrelated (R = 0.37). Since we hypothesized that protection against MMS involved stabilization of membranes, the capacity of the 14 compounds to stabilize membranes in an unrelated system (i.e. prevention of erythrocyte osmotic rupture) was assayed. Chemoprotection against both MNNG and MMS correlated with reduced RBC fragility (R = 0.97 and 0.70, respectively). One of the better protecting compounds, 4b,5,9b,10-tetrahydroindeno[1,2-b]indole, was also protective against hepatocellular toxicity mediated by acetaminophen, carbon tetrachloride and tert-butyl hydroperoxide, suggesting a fundamental basis in the mechanism of chemoprotection. We propose that methylating agents and perhaps other chemical toxicants destabilize cellular membranes resulting in hepatocellular injury. For MNNG, radical mediated events may result in membrane destabilization; for MMS, membranes are destabilized without concurrent radical events. The current studies provide a basis for future work to determine structure-activity relationships of chemoprotective agents, examine protection mechanisms, and develop better protective compounds.

Animals↗
PubMed1991

Intrinsic acute toxicity and hepatic enzyme inducing properties of the chemoprotectants indole-3-carbinol and 5,10-dihydroindeno[1,2-b]indole in mice.

Indole-3-carbinol (I-3-C) and 5,10-dihydroindeno[1,2-b]indole (DHII) have been shown to be protective against carbon tetrachloride and other chemicals that cause hepatic toxicity. In part, this protection appears to be afforded by the ability of these compounds to act as antioxidants, with DHII having much the greater efficacy. In order to understand the mechanisms of chemoprotection, as well as the potential for therapeutic and pharmaceutical use in humans, the antioxidants I-3-C and DHII were examined for their intrinsic acute toxicity, and their hepatic enzyme inducing properties in mice. The results were compared with those of the well characterized agent phenobarbital. Following treatment by gavage for 10 days with 50 mg compound/kg body weight, I-3-C produced modest (10-50%) increases in hepatic cytochrome P-450, aminopyrine N-demethylase, UDP-glucuronosyl transferase (UDPGT) and glutathione S-transferase (GST), and a four-fold increase in NAD(P)H: (quinone acceptor) oxidoreductase (quinone reductase) activity. DHII did not alter oxidative enzyme activities, but increased GST and UDPGT by about 50%, and quinone reductase over five-fold. In the acute toxicity studies, DHII produced no observable 24-hr acute toxicity up to 4 g/kg body weight, except for a slight decrease in haematocrit. However, I-3-C exhibited a dose-dependent toxicity above 100 mg/kg body weight, including a decrease in hepatic reduced glutathione after 2 hr and severe neurological toxicity, and the release of liver enzymes to the plasma at 24 hr. We conclude, on the basis of the superior antioxidation efficacy of DHII, its enzyme-inducing properties, and intrinsic toxicity, that DHII or cogeners thereof have great potential as chemoprotective or therapeutic agents. However, I-3-C does not have such potential.

Animals↗
PubMed1991

Chemoprotective and hepatic enzyme induction properties of indole and indenoindole antioxidants in rats.

Three indole antioxidants were compared for their efficacy to inhibit lipid peroxidation, prevent chemical hepatotoxicity and induce enzyme systems involved in the biotransformation of xenobiotics. The dietary indolyl compound indole-3-carbinol (I-3-C), and the synthetic compounds 5,10-dihydroindeno[1,2-b]-indole (DHII) and 4b,5,9b,10-tetrahydroindeno[1,2-b]indole (THII) inhibited carbon tetrachloride (CCl4)-initiated lipid peroxidation in rat-liver microsomes, with the order of efficacy THII greater than DHII = butylated hydroxytoluene (BHT) much greater than I-3-C. Each of the indole compounds protected isolated rat hepatocytes against toxicity by CCl4, N-methyl-N'-nitro-N-nitrosoguanidine and methylmethanesulphonate (THII congruent to DHII much greater than I-3-C). In vivo administration of the indole compounds 1 hr before treatment with CCl4 protected against hepatotoxicity (THII greater than DHII greater than I-3-C). For the enzyme induction studies, phenobarbital and beta-naphthoflavone were used as standards, with corn-oil vehicle controls. The compounds were administered by gavage at 50 mg/kg body weight/day for 10 days. I-3-C produced increases in levels of hepatic cytochromes P-450 and ethoxyresorufin O-deethylase (EROD) activity, as well as in UDP-glucuronosyl transferase (UDPGT), glutathione S-transferase (GST), glutathione reductase (GSSG-Red) and quinone reductase. I-3-C produced decreased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities. DHII produced increases in EROD, UDPGT, GST, GSSG-Red and quinone reductase, with decreases in NDMA-demethylase and GSH-Px activities. The only observed effect of THII was a modest induction of EROD activity. After treatment with the indole compounds for 10 days, I-3-C enhanced, while DHII diminished, CCl4-mediated 24-hr hepatotoxicity in rats. We conclude that DHII and THII are suitable candidates to develop further as potential chemoprotective and therapeutic agents for use in humans to treat disorders involving free radicals. THII has the greater radical scavenging efficacy, whereas DHII has the greater capacity to induce many different antioxidative enzymes.

Administration, Oral↗
PubMed1979

Anaerobic rat heart: mitochondrial role in calcium uptake and contractility.

In order to evaluate the manner by which fumarate enhances contractility in the anaerobic heart, we examined Ca++ movements in isolated heart mitochondria and in the isolated perfused heart. Our experiments showed that in isolated antimycin A plus cyanide treated mitochondria: (a) Ca++ uptake was promoted by electron transport generated by fumarate-dependent oxidation of NADH, (b) Ca++ stimulated fumarate-dependent oxidation of NADH, (c) the ratio of Ca++ uptake:NADH oxidized was 1.7 and (d) the Ca++ sequestered is transiently highly mobile and is rapidly released upon collapse of the membrane potential. In anaerobic hearts perfused with glucose plus fumarate, malate and glutamate Ca++ levels were the same as those in oxygenated hearts while in anaerobic organs perfused with or without glucose Ca++ content was appreciably lower. Succinate production in anaerobic heart perfusions was related to: (a) an increased retention of Ca++ by the heart, (b) a diminution in peak aortic pressure generated by cardiac contractions and (c) an increase in heart rate. The information obtained indicates that mitochondria have a capability for Ca++ movement which be used physiologically, particularly in fumarate perfused anaerobic hearts, to assist the mechanism for contraction and relaxation of the heart.

Adenosine Diphosphate↗
PubMed1976

Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria.

1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.

Animals↗