Biomedical subjects

B M Hasspieler

Publications and source records attributed to B M Hasspieler.

10 recordsLinked to original sources

PubMed1997

Roles of DT diaphorase in the genotoxicity of nitroaromatic compounds in human and fish cell lines.

The genotoxicity of nitroaromatic compounds was examined in two cultured cell lines, namely, a human hepatoma cell line, HepG2, and a brown bullhead fibroblast cell line, BB. Furthermore, the role of the quinone-reducing enzyme DT diaphorase [NAD(P)H:(quinone acceptor) oxidoreductase] was examined with respect to its influence on the genotoxic effects of model nitroaromatic pollutants. The nitroreductive characteristics of these two cell lines were examined using an acetylated cytochrome c reduction assay for enzymatic nitroreductase activity. Subsequently, the influence of DT diaphorase on the genotoxicity of two model nitroaromatics, 4-nitroquinoline 1-oxide (4NQ) and nitrofurantoin (NF), revealed that DT diaphorase was the predominant 4NQ reductase in cytosols of both cell lines, but played a lesser role in NF reduction in both species. Despite these interspecific similarities, results revealed marked qualitative differences between the two species in terms of the influence of DT diaphorase on quinone-mediated genotoxicity. When pretreated with the DT diaphorase inhibitor dicoumarol, HepG2 cells exhibited an exacerbation of genotoxicity in the presence of 4NQ, indicating a protective influence of the enzyme. In contrast, 4NQ genotoxicity in BB cells was reduced in the presence of dicoumarol, indicating a deleterious effect of DT diaphorase activity. Conversely, dicoumarol pretreatment was moderately protective against NF-mediated genotoxicity in HepG2 cells but exacerbated NF toxicity in BB cells. This study illustrates the manner in which functionally analogous enzymes may have markedly distinct influences on xenobiotic toxicity in different cellular systems.

4-Nitroquinoline-1-oxide↗

PubMed1996

Influence of DT diaphorase on quinone-mediated genotoxicity in human and fish cell lines.

The influence of the quinone-reducing enzyme, DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase], on the genotoxicity of quinones was examined in two cell lines, namely a human hepatoma cell line, HepG2 and a brown bullhead fibroblast cell line, BB. The quinone-reductive characteristics of these two cell lines were examined using an acetylated cytochrome c reduction assay for enzymatic reductase activity. Subsequently, the influence of DT diaphorase on the genotoxicity of two model quinones, menadione (MND) and 9,10-phenanthrenequinone (PQ) was examined in an alkaline unwinding assay for DNA single-strand breaks. Results revealed that DT diaphorase was the predominant quinone reductase in cytosols of both cell lines, and that levels of specific DT diaphorase activity were generally equivalent in the two species. Despite these similarities, results revealed marked qualitative differences between the two species in terms of the influence of DT diaphorase on quinone-mediated genotoxicity. When pretreated with the DT diaphorase inhibitor, dicoumarol, HepG2 cells exhibited a marked exacerbation of genotoxicity in the presence of either MND or PQ, indicating protective influence of the enzyme. In contrast, quinone genotoxicity in BB cells was not affected by DT diaphorase inhibition, indicating the lack of a protective effect of DT diaphorase. This study illustrates the manner in which functionally analogous enzymes may have markedly distinct influences on xenobiotic toxicity in different cellular systems.

Animals↗

PubMed1995

Human bioassays to assess environmental genotoxicity: development of a DNA break bioassay in HepG2 cells.

OBJECTIVES: Increasing interest in environmental health issues has created a demand for improved methods for the assessment of pollutant effects on humans. Our laboratory has developed an in vitro assay for the quantification of genotoxicity, monitored as DNA single strand breaks (SSB), in the HepG2 human hepatoma cell line. DESIGN AND METHODS: This assay procedure, which is based upon alkaline unwinding and hydroxylapatite DNA chromatography, is both rapid and simple to perform. RESULTS: HepG2 cells responded to the standard mutagen, 4-nitroquinoline N-oxide, demonstrating SSB formation at concentrations above 0.1 mumol/L. Phenanthrene-9,10-quinone, a component of diesel exhaust, mediated SSB formation at concentrations above 250 nmol/L. Finally, an extract of contaminated sediment from the Great Lakes Basin mediated SSB formation in a dose-dependent manner. CONCLUSIONS: These results illustrate the utility of this human genotoxicity assay for future use in screening of environmental pollutants.

4-Nitroquinoline-1-oxide↗

PubMed1994

Glutathione-dependent defense in channel catfish (Ictalurus punctatus) and brown bullhead (Ameriurus nebulosus).

Glutathione-dependent defense against xenobiotic toxicity is a multifaceted phenomenon that has been well characterized in mammals. This study undertakes a comparison of two benthic fish species, the channel catfish and brown bullhead, in terms of characteristics of the glutathione system. The channel catfish, a species that has seldom been observed to express pollutant-mediated neoplasia in field studies, was observed to have significantly higher constitutive levels of hepatic total glutathione and reduced glutatione (GSH). Brown bullhead, a species that is often observed to express neoplasia in contaminated systems, had significantly higher hepatic levels of glutathione disulfide. Furthermore, catfish expressed higher levels of activity of the enzymes gamma-glutamylcysteine synthetase (GCS), glutathione reductase (GR), and glutathione S-transferase, whereas bullhead expressed higher hepatic glutathione peroxidase (GPOX) activity. Both species responded to treatment with the redox active quinone, menadione, by expressing elevated hepatic content of total glutathione. However, the induction response was more rapid and more extensive in catfish compared to that in bullhead. This is attributable to the observed interspecific difference in GCS activity. Following treatment with the organic peroxide, tert-butyl hydroperoxide (t-BOOH), bullhead hepatic glutathione was depleted up to 4 hr post-treatment, whereas catfish demonstrated no significant depletion of glutathione in response to t-BOOH. The differing responses to t-BOOH are attributable to interspecific differences in hepatic GPOX and GR activity. Bullhead, therefore, appear to be more susceptible to the effects of GSH arylators and oxidants based upon constitutive levels of glutathione, related enzyme activities, and the response of this system to model xenobiotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Dicoumarol-sensitive NADPH: phenanthrenequinone oxidoreductase in channel catfish (Ictalurus punctatus).

Phenanthrenequinone (PQ), which occurs widely as a pollutant and as a major metabolite of phenanthrene in a number of species, has been demonstrated to undergo futile redox cycling leading to oxidative stress. In the presence of cytosolic fractions of selected channel catfish tissues, PQ undergoes enzymatic reduction which is mediated by either NADH or NADPH and is composed of dicoumarol-sensitive and -insensitive components. Most notably, gastric cytosol catalyzed a disproportionately high level of NADPH-dependent, dicoumarol-sensitive PQ reduction as compared to gill, liver, and kidney cytosols. In the presence of stomach cytosol and NADPH, PQ facilitated production of superoxide anion at rates several fold higher than those mediated by menadione. The dicoumarol-sensitive PQ-reducing agent, which we have termed NADPH: phenanthrenequinone oxidoreductase (PQR), was purified by affinity chromatography and was demonstrated to be separable from DT diaphorase activity in gastric cytosol. Under aerobic conditions, purified PQR facilitates redox cycling of PQ as indicated by continued NADPH oxidation and hydrogen peroxide production. Under anaerobic conditions, NADPH oxidation is limited to a quantity indicative of PQ reduction to the hydroquinone. Substrate specificities, pH profiles, and kinetic characteristics combine to indicate that PQR represents a novel quinone reductase in this species.

Animals↗

PubMed1994

Human bioassays to assess environmental genotoxicity: development of a DNA repair assay in HepG2 cells.

A direct assessment of the effects of environmental chemicals on human health has been hampered by the lack of suitable experimental systems. We have recently employed a human liver cell line (HepG2) to assess the biological effects of pollutants at both cellular and DNA levels. A Neutral Red dye uptake assay was used to assess potential cytotoxic effects of xenobiotics. DNA damage was quantified using an unscheduled DNA synthesis assay that measures repair that is induced following exposure to genotoxic compounds. HepG2 cells responded to the known mutagens, 4-nitroquinoline N-oxide and methylmethane sulfonate, both in the Neutral Red assay for cytotoxicity and two DNA repair assays for genotoxicity (monitored autoradiographically or by liquid scintillation counting). The HepG2 DNA repair and cytotoxicity assays also responded to an extract (containing polycyclic aromatic hydrocarbons) of sediment obtained from a polluted site in the Great Lakes. Results indicate that this system can be deployed further to assess potential cyto- and genotoxicity of pollutants. The development of human cell culture assays is a critical step towards a full assessment of the risk that such pollutants pose to human health.

4-Nitroquinoline-1-oxide↗

PubMed1992

Effects of buthionine sulfoximine and diethyl maleate on glutathione turnover in the channel catfish.

Despite the growing use of fish in toxicological studies, little is known regarding glutathione (GSH) metabolism and turnover in these aquatic species. Therefore, we examined GSH metabolism in the liver and gills of channel catfish (Ictalurus punctatus), a commonly employed aquatic toxicological model. Treatment of channel catfish with L-buthionine-S,R-sulfoximine (BSO, 400 or 1000 mg/kg, i.p.), an inhibitor of GSH biosynthesis, did not deplete hepatic GSH in channel catfish. In addition, hepatic GSH concentrations did not fluctuate in catfish starved for 3 days, indicating relatively slow turnover of hepatic GSH. However, hepatic GSH concentrations were reduced significantly (P less than 0.05) after 7 days of starvation. Administration of the thiol alkylating agent diethyl maleate (DEM, 0.6 mL/kg, i.p.) resulted in depletion of 85% of hepatic GSH at 6 hr post-DEM, with complete GSH recovery observed at 24 hr post-DEM. Co-administration of BSO and DEM (1000 mg/kg, 0.6 mL/kg, respectively) substantially depleted gill GSH and eliminated detectable liver GSH. Following BSO/DEM, GSH recovery in hepatic mitochondria occurred more rapidly than did liver cytosolic GSH. gamma-Glutamylcysteine synthetase (GCS) activities were comparable in the 10,000 g supernatants of catfish liver and gills (204 +/- 21 and 268 +/- 20 nmol/min/mg protein, respectively) whereas gamma-glutamyltranspeptidase (GGT) activity was not detected in the 600 g post-nuclear fraction of either liver or gills. In conclusion, i.p. administration of DEM was an effective means for achieving short-term hepatic GSH depletion in channel catfish, whereas co-administration of BSO and DEM elicited prolonged and extensive hepatic GSH depletion in this species. Like rodents, channel catfish maintained physiologically distinct hepatic mitochondrial and cytosolic GSH pools, and also regulated hepatic GSH levels by in situ hepatic GSH biosynthesis. However, unlike rodents, there was no evidence for a labile hepatic cytosolic GSH pool in channel catfish. These similarities and differences need to be considered when designing toxicological studies involving the GSH pathway in channel catfish and possibly other fish species.

Animals↗

PubMed1992

DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase] facilitates redox cycling of menadione in channel catfish (Ictalurus punctatus) cytosol.

Characteristics of DT diaphorase (NAD(P)H: (quinone acceptor) oxidoreductase, DTD) activity in Ictalurus punctatus and the effect of DTD activity on menadione (MND)-mediated reduction of acetylated cytochrome c (AcC) were examined. DTD activity in cytosols of four organs followed a distinct gradient in the order stomach greater than gill greater than liver greater than posterior kidney. A similar gradient was observed in organ-specific rates of in vitro AcC reduction in the presence of either NADH or NADPH as reducing equivalent. A greater proportion of the AcC reduction rate was sensitive to inhibition by dicoumarol (DC) in organs with relatively high DTD specific activity (e.g., stomach) than in organs with low DTD activity (e.g., kidney). No such trend was observed in the superoxide dismutase (SOD)-sensitive proportion of AcC reduction rates. DTD was observed to contribute to MND-mediated superoxide production to a greater extent in organs with high DTD activity than in organs with low DTD activity. DC-sensitive (i.e., DTD-mediated) AcC reduction was observed to increase with organ-specific DTD activity, and the majority of the AcC reduction rate was inhibitable by SOD. These findings demonstrate a direct contribution by DTD activity to MND-mediated superoxide production in this in vitro system. The role of I. punctatus DTD as a possible deleterious agent in quinone metabolism and implications regarding the traditional conception of DTD as a detoxifying enzyme are discussed.

Animals↗

PubMed1988

Toxicity, localization and elimination of the phototoxin, alpha-terthienyl, in mosquito larvae.

Mosquito larvae were examined to determine interspecific and interstrain differences in susceptibility to the larvicidal effects of the plant-derived phototoxin, alpha-terthienyl (alpha-T). The LC50 values were as follows: Aedes aegypti, 4 ppb; Ae. epactius, 6 ppb; anopheles stephensi, 14 ppb; malathion-susceptible Culex tarsalis (S), 12 ppb; malathion-resistant Cx. tarsalis (R), 16 ppb. Fluorescence studies indicated localization of alpha-T in the midgut epithelium and in the lumen of Malpighian tubules. Rates of elimination of tritiated alpha-T differed significantly between Ae. aegypti and Cx. tarsalis (S) larvae. Rate of 3H-alpha-T elimination was inversely correlated with susceptibility to the toxic effects of the compound. The toxicological significance of selective alpha-T accumulation and the importance of alpha-T elimination in determining sensitivity are discussed.

Aedes↗

PubMedDate not supplied

In vitro toxicological methods for environmental health testing.

Increasing public interest in environmental health issues has created a demand for alternatives to using animals for assessing the toxic effects of chemical mixtures on humans. This review focuses on applications of in vitro toxicological screening methods developed for human health biomonitoring using cultured clonal cell lines, which have the which have the following advantages: genetic variation between samples and experiments is minimal; the cultivation of cell lines is rapid and consistent conditions for culture are easily maintained; most of the phenotypic variation that is encountered with use of cell donors is eliminated; and radiolabeled precursors can be used for labeling and quantifying protein and DNA. We describe the current state of development of in vitro toxicity testing methods, present detailed procedures for the test methods optimized in our laboratory, and compare these techniques with other approaches. Toxicity testing using cell lines provides a mechanism to quantify the risks associated with environmental exposure to chemical mixtures.

Animal Testing Alternatives↗