Biomedical subjects

J K Chipman

Publications and source records attributed to J K Chipman.

At least 37 records · Page 2Linked to original sources

PubMed1999

EGF-Induced receptor autophosphorylation in primary hepatocytes isolated from phenobarbitone-treated mice.

Phenobarbitone (PB) treatment of mice causes a decrease in the growth factor responsiveness of hepatocytes. Here, epidermal growth factor receptor (EGFR) expression and receptor autophosphorylation was determined in hepatocytes isolated from control and PB-treated mice. There was a decrease in the level of EGFR expression in hepatocytes isolated from mice following PB administration when compared to controls. EGF caused an approximate 20-fold increase of the 170 kD phosphotyrosine band in control hepatocytes, which was inhibited by the EGFR specific tyrosine kinase inhibitor 4, 5-dianilinopthalamide. Following PB treatment, the degree of basal receptor phosphorylation (in the absence of EGF) was significantly greater and therefore the fold rise in EGFR phosphorylation in isolated hepatocytes was lower than in controls. However, the overall extent of EGF-induced receptor phosphorylation was not diminished in hepatocytes isolated from PB-treated mice. Therefore the reduction in responsiveness to growth factors seen in hepatocytes ex vivo or the cessation of proliferation observed in vivo following PB administration is unlikely to be attributed to a decrease in ligand binding and subsequent receptor autophosphorylation.

Animals↗

PubMed1999

Antioxidant effects of isoflavonoids and lignans, and protection against DNA oxidation.

The antioxidant capability of a series of isoflavonoid and lignan compounds in both cellular and cell-free systems has been investigated, and related to structure. Nordihydroguaiaretic acid exhibited a potent antioxidant activity in both HepG2 and MDA-MB-468 cells (IC50 5.3 and 1.1 microM respectively), as determined by inhibition of 2',7'-dichlorofluorescin oxidation via t-BOOH, although no inhibition was observed with other compounds tested in this system. All compounds inhibited the formation of 8-oxodeoxyguanosine in DNA exposed to hydroxyl radicals via gamma irradiation or the Fenton reaction. Whilst almost complete inhibition of gamma irradiation-induced damage was achieved (IC50 ranged from 0.2 to 0.8 microM), inhibition was less pronounced with the Fenton system. The ability of all compounds to interact with DNA (as well as with reactive oxygen and iron) was also demonstrated by scanning UV spectroscopy, suggesting that the compounds may inhibit DNA oxidation at least in part by binding to DNA. Hydroxyl radical-scavenging, iron-chelating and DNA-binding activity of these compounds supports their proposed role as natural cancer-protective agents.

Antioxidants↗

PubMed1999

ANIT-induced disruption of biliary function in rat hepatocyte couplets.

alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rats, involving damage to biliary epithelial cells; our study aims to investigate whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatocyte couplets were used to investigate biliary function in vitro by canalicular vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), within the canalicular vacuole between the two cells. After a 2-h exposure to ANIT, there was a concentration-dependent inhibition of cVA (cVA-IC50; 25 microM), but no cytotoxicity (LDH leakage or [ATP] decline) within this ANIT concentration range. There was no loss of cellular [GSH] at low ANIT concentrations, but, at 50 microM ANIT, a small but significant loss of [GSH] had occurred. Diethylmaleate (DEM) partially depleted cellular [GSH], but addition of 10 microM ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANIT to these cells reduced cVA further, but the magnitude of this further reduction was no greater than that caused by ANIT alone, indicating that glutathione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphological change in the canalicular vacuoles but only a small, non-significant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the pericanalicular web. The results are in accord with a disruption of hepatocyte canalicular secretion within two h in vitro, at low, non-cytotoxic concentrations of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect.

1-Naphthylisothiocyanate↗

PubMed1998

Production of DNA strand breaks by N-nitrosodimethylamine and 2-amino-3-methylimidazo[4,5-f]quinoline in THLE cells expressing human CYP isoenzymes and inhibition by sulforaphane.

The production of DNA strand breaks by N-nitrosodimethylamine (NDMA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been observed in T5-2E1 (expressing human CYP2E1) and T5-1A2 (expressing human CYP1A2) human liver cells respectively, using the Comet assay. Responses were statistically significant (P<0.05) and concentration dependent (0.01-1 microg ml-1 NDMA and 0.1-10 microg ml-1 IQ) and were not observed in T5-neo cells devoid of cytochrome P450 activity. Sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) (0.1-10 microM) gave a marked inhibition of DNA strand breakage by these carcinogens (P<0.05 linear regression). This was seen in the absence of cytotoxicity and in the absence of an inhibition of H2O2-induced DNA strand breakage. The ability of sulforaphane to inhibit both CYP2E1 and CYP1A2-mediated genotoxicity therefore is relevant to human isoforms of these enzymes and may contribute to a chemopreventative activity.

Antimutagenic Agents↗

PubMed1998

DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring.

This review considers the potential for DNA strand breaks, particularly as measured by the comet assay, to act as a biomarker of genetic toxicity in fish and other aquatic species. The background need for such biomarkers is introduced in relation to carcinogenicity, reproductive effects and other adverse effects of pollution. Sensitive measurements of DNA strand breakage can be achieved, e.g., by alkaline elution, alkaline unwinding or by single cell gel electrophoresis (comet) techniques. The DNA damage can be a reflection, not only of direct strand breakage, but also of alkali-labile sites and of repair enzyme-mediated breakage (i.e., is non-specific). A range of genotoxic chemicals (both with and without the requirement for metabolic activation) give positive effects in various cell types of vertebrate and invertebrate aquatic species, following in vitro and in vivo exposures under laboratory conditions. A limited number of analyses of organisms exposed to polluted waters or sediments in the field have implicated DNA strand breakage as a relatively sensitive, rapid and broad specificity indicator of genotoxic pollutant exposure. The comet assay deserves further exploitation to assess inter-individual and inter-cell variability in response to pollutants and naturally occurring genotoxic stimuli, and to assess the persistence of these effects.

Animals↗

PubMed1998

DNA oxidation by potassium bromate; a direct mechanism or linked to lipid peroxidation?

Following incubation of calf thymus DNA with potassium bromate (KBrO3) and glutathione (GSH), a statistically significant increase in the concentration of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine was measured. This was GSH-dependent and was associated with loss of GSH during incubation. In contrast, 8-oxodG was not found to be elevated significantly in either total tissue DNA or mitochondrial DNA isolated from Sprague-Dawley rat kidney perfused in situ with KBrO3 (5 mM) for 15 min or 1 h. There was also no associated increase in the level of renal lipid peroxidation or reduced or oxidised GSH. Following intraperitoneal administration of KBrO3 to Sprague-Dawley rats, a dose of 100 mg/kg (maximum tolerated) gave evidence for oxidative stress in the kidney at 24 h as indicated by a significant increase in lipid peroxidation (P < 0.05) and oxidised GSH (P < 0.05). This was associated with a greater than 2-fold, significant (P < 0.01) increase in the level of 8-oxodG in kidney total DNA and a 57% (not statistically significant) increase in kidney mitochondrial 8-oxodG. Pretreatment of rats with diethylmaleate (DEM) to deplete GSH, elevated the toxicity of 100 mg/kg KBrO3. However, at a dose of 20 mg/kg, no change in any of the parameters indicative of kidney oxidative stress (including indicators of oxidative DNA damage; 8-oxodG or etheno-DNA adducts, which can be produced by lipid peroxides) was seen either with or without DEM pretreatment with the exception of a small but statistically significant (P < 0.05) increase in mitochondrial 8-oxodG when KBrO3 was given following DEM pretreatment. DNA oxidation in the kidney is therefore not inhibited by GSH depletion (contrasting with in vitro findings) and requires a sustained exposure at a near-toxic concentration of KBrO3 which is associated with lipid peroxidation and GSH oxidation. The results do not support a role, in rat kidney, of a direct, GSH-mediated mechanism for KBrO3-induced DNA oxidation as seen in vitro.

Animals↗

PubMed1998

Detection of DNA strand breaks in isolated mussel (Mytilus edulis L. ) digestive gland cells using the "Comet" assay.

Isolated mussel (Mytilus edulis L.) digestive gland cells were analyzed using the single-cell gel electrophoresis or "comet" assay to assess the ability of potential aquatic contaminants to induce DNA strand breaks (SBs) and to investigate the potential application of this technique as part of an aquatic biomonitoring regime. Freshly prepared cell suspensions from digestive gland were exposed in vitro to hydrogen peroxide (H2O2, 0-200 microM), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX, 0-200 microM), benzo[a]pyrene (BaP, 0-200 microM), 1-nitropyrene (1-NP, 0-250 microM) and nitrofurantoin (NF, 0-1000 microM) for 1 h in the dark at 15 degreesC in the presence of the DNA repair inhibitor cytosine-beta-D-arabinofuranoside (araC). DNA strand breakage was measured using the comet assay. There were significant concentration-dependent increases in the percentage of DNA in the comet tail (mean values+/-SD) for all doses compared with controls (P<0.05) with H2O2 (up to 61.4+/-5.1% at 100 microM), MX (up to 34. 3+/-2.2% at 200 microM), BaP (up to 24.7+/-5.1 at 100 microM), 1-NP (up to 54.7+/-5.0% at 200 microM), and NF (up to 68.1+/-4.5% at 500 microM). There was a decrease (P<0.05) in viability (eosin Y exclusion) of exposed compared with control cells at 200 microM H2O2 and BaP only. This study has demonstrated the potential of the comet assay to detect DNA strand breakage at subcytotoxic concentrations of a range of agents, some of which require metabolic activation. This may provide a sensitive, but nonspecific, molecular biomarker of genotoxicity.

Animals↗

PubMed1998

The rodent nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin inhibits intercellular communication in rat but not guinea-pig hepatocytes, perturbing S-phase but not apoptosis.

Previously, we have shown that the peroxisome proliferator (PP), nafenopin, induces S-phase in rat hepatocytes and suppresses apoptosis in hepatocytes from both rat and guinea-pig. Here, we confirm and extend these findings by defining the time course of growth perturbation and by correlating this with species differences in loss of gap junctional intercellular communication (GJIC). GJIC is associated with nongenotoxic carcinogenesis, possibly reflecting a tumour suppresser role of the connexins. Fluorescence microscopy of Hoechst 33258-stained rat or guinea-pig hepatocyte monolayers showed 1% apoptosis during the first 8 h of culture, peaking to 2-2.5% at 20-24 h. Nafenopin suppressed apoptosis compared with controls in both rat and guinea-pig, measured at 20 h and 24 h onwards, respectively. The induction of S-phase in rat hepatocytes by nafenopin could be detected as early as 4 h after compound addition whereas S-phase was not altered by nafenopin in guinea-pig hepatocytes. Intercellular communication as measured by intercellular transfer of microinjected Lucifer Yellow CH was observed during the first 14 h of primary rat hepatocyte culture peaking at a maximum value of 88 +/- 3.0% after 7 h. In hepatocyte cultures from guinea-pig, dye-coupling levels were maintained between 88 +/- 3.0 and 93 +/- 3.0% within 2-10 h of culture and by 12 h showed only a slight decrease to 72 +/- 3.0%. In the rat, significant inhibition was observed at 4 h after administration of nafenopin since GJIC was reduced by 20 +/- 5% compared with vehicle control. By contrast, in the presence of nafenopin, the level of dye-coupling between guinea-pig hepatocytes did not decrease but remained between 85 +/- 5 and 93 +/- 3.0%, similar to that observed in control guinea-pig cultures. The data obtained contribute to our understanding of the role of GJIC inhibition in the perturbation of cell survival and proliferation caused by nongenotoxic hepatocarcinogens.

Animals↗

PubMed1998

Inhibition of mouse and human CYP 1A- and 2E1-dependent substrate metabolism by the isoflavonoids genistein and equol.

The inhibitory effect of the isoflavonoids genistein and equol on cytochrome P450 activities has been investigated. Genistein and equol inhibited the high capacity component of p-nitrophenol (CYP2E1 substrate) metabolism in liver microsomes from acetone-induced mice with IC50 values of approximately 10 mM and 560 microM, respectively (cf. diethyldithiocarbamate, IC50, 69 microM). Using human CYP2E1 from a specific expression system (which overcame multienzyme involvement in the rodent system), non-competitive inhibition was also seen with both isoflavonoids. Genistein and equol also inhibited the high capacity component of ethoxyresorufin (CYP1A substrate) metabolism in liver microsomes from beta-naphthoflavone-induced mice with IC50 values of 5.6 mM and 1.7 mM, respectively (cf. alpha-naphthoflavone, IC50 0.8 microM). Using human CYPIA2 from a specific expression system, noncompetitive inhibition was seen with both isoflavonoids. CYP1A1 inhibition offers a possible explanation for the chemopreventative effect of genistein against, for example, dimethylbenz[a]anthracene genotoxicity reported in animals but the IC50 values negate the relevance of this specific chemopreventative action at the levels likely to be achieved from the human diet.

Animals↗

PubMed1998

Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells.

Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.

Breast Neoplasms↗

PubMed1998

Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-kappaB DNA binding activity and DNA damage in HepG2 cells.

We have investigated the effect of the plant-derived flavonoid quercetin in relation to potential oxidant and antioxidant activity on nuclear factor kappaB (NF-kappaB) binding activity and DNA integrity in HepG2 cells. Gel mobility shift assays using a gamma-32P-labelled NF-kappaB oligonucleotide probe showed that treatment of HepG2 cells with quercetin (up to 10 microM, sub-cytotoxic) did not elevate NF-kappaB binding activity of nuclear extract protein but did inhibit binding activity of an extract from cells treated with the oxidant H2O2. A similar inhibition by quercetin of H2O2-induced NF-kappaB transcriptional activation was demonstrated using a cat reporter gene assay. Considering oxidative DNA damage, using single cell gel electrophoresis (comet) assay we have demonstrated that quercetin (10 microM and below) did not induce DNA strand breaks. However, a marked and statistically significant (P < 0.01 at 10 microM) inhibition of strand breakage produced by H2O2 was detected. The specific formation of 8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA exposed to either gamma-irradiation or the Fenton reaction system was also inhibited (P < 0.01 at 10 microM) by quercetin in a dose-dependent manner. This was not accompanied by formation of 8-oxodG by quercetin itself. The inhibition of 8-oxodG formation by gamma-irradiation was more potent (IC50 = 0.05 microM) than that by the Fenton reaction (IC50 = 0.5 microM), implying that the mechanism of protection may be different between the two systems. The inhibition of both NF-kappaB binding activity and oxidative DNA damage suggests that its antioxidant potential outweighs its oxidative potential in a cellular environment, which may contribute to anticarcinogenic and anti-inflammatory effects.

Antioxidants↗

PubMed1997

Production of unscheduled DNA synthesis in rodent hepatocytes in vitro, but not in vivo, by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX).

Incubation of both rat and mouse hepatocytes with 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in vitro resulted in a dose-dependent increase in unscheduled DNA synthesis (UDS) at sub-cytotoxic concentrations (1-10 microM MX; 20 h incubation). Depletion of glutathione stores by pre-treatment of rat hepatocytes with buthionine sulfoximine did not result in a significant increase in UDS produced by MX. In contrast, MX did not induce UDS in mouse hepatocytes ex vivo either 3 or 16 h following administration of a single oral dose of 100 mg/kg MX. Despite the ability of MX to produce repairable DNA damage, restricted access of MX to the liver may prevent a measurable UDS response in vivo.

Administration, Oral↗

PubMed1997

The response of hepatocytes isolated from phenobarbitone treated mice to mitogenic growth factors.

The ability was investigated of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) to stimulate DNA synthesis in hepatocytes isolated from C57B1/6J mice following 1, 3, 7, 30 and 90 days pre-treatment with the hepatomegalic drug, phenobarbitone (PB). A 3-fold increase in S-phase labelled hepatocytes was observed in the absence of growth factors after 3 days treatment with PB, which was not seen at other investigated time points. This suggests that the proliferative influence present in vivo at this time interval is maintained in the ex vivo model. Maximum labelling indices of > 5-fold the unstimulated control value were observed in hepatocytes isolated from control and 1 day PB pre-treated mice when cultured in the presence of 5 or 10 ng/ml EGF or HGF. Hepatocytes isolated from 3, 7, 30 or 90 day treated mice showed a considerably reduced responsiveness to growth factors; maximum labelling indices did not exceed by a factor of 2 the value obtained in the absence of growth factors. However, the apparent decrease in responsiveness to growth factors in hepatocytes isolated from 3 day pre-treated mice was due to an increased background level of proliferation and the attainment of a 'ceiling level' of DNA synthesis at approx. 35%. DNA synthesis was not further enhanced by addition of both EGF and HGF. This maximal level of stimulation may indicate that only a specific hepatocyte sub-population is capable of responding to growth factors under the conditions employed. The loss in sensitivity to mitogenic stimuli after 7 days PB pre-treatment correlates with a reported decrease in receptor protein and mRNA levels in rats and coincides with the in vivo shift from hyperplasia to hypertrophy.

Animals↗

PubMed1997

The isoflavones equol and genistein do not induce xenobiotic-metabolizing enzymes in mouse and in human cells.

1. In view of the anticarcinogenic effects of the isoflavonoid genistein and the known ability of various flavonoids to induce carcinogen-metabolizing enzymes, the ability of the isoflavonoids genistein and equol to induce these enzymes was studied in mouse. 2. In comparison with induction by the positive control beta-naphthoflavone (40 mg/kg i.p. 4 days) neither genistein or equol (0.4-40 mg/kg i.p. 4 days) gave a significant induction of ethoxyresorufin O-deethylase, p-nitrophenol oxidase or immunoreactive CYP1A2 or CYP2E1. There was also no induction of erythromycin-N-demethylase or elevation of immunoreactive CYP3A1. 3. In contrast with the induction by beta-naphthoflavone of glutathione S-transferase protein and activity towards 1-chloro-2,4-dinitrobenzene, neither isoflavone exhibited such induction. 4. Response elements for human CYP1A1, glutathione S-transferase lambda a and the xenobiotic response element (XRE) in HepG2 cells were activated by beta-naphthoflavone but not by genistein or equol. 5. The isoflavones have been found not to induce a range of enzymes involved in carcinogen metabolism. The lack of enzyme induction differs from the characteristics of many other flavonoids. The results do not support the monofunctional induction of GST as a basis of anticarcinogenicity.

Animals↗

PubMed1997

Effects of quercetin on drug metabolizing enzymes and oxidation of 2',7-dichlorofluorescin in HepG2 cells.

1. The effects of quercetin on drug metabolising enzymes and oxygen radicals were studied in human HepG2 cells. 2. Cytotoxicity of quercetin in HepG2 cells was seen at 50 microM and above as evaluated by lactate dehydrogenase (LDH) leakage, neutral red (NR) uptake, and 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. 3. Quercetin inhibited activity of human cytochrome P-450 towards ethoxycoumarin and ethylresorufin at relatively low substrate concentrations (0.1 microM and above). 4. In comparison to induction by the positive control (beta-naphthoflavone; 1.0 microM), quercetin did not significantly induce the metabolism of ethoxycoumarin or glutathione-S-transferase (GST) protein or activity. 5. Response elements for human CYP1A1, GST lambda a, xenobiotic response element (XRE), fos, HSP70, CRE, p53, NF kappa B and DNA damage (GADD) in HepG2 cells were not activated by quercetin. 6. Quercetin exhibited antioxidant activity in HepG2 cells as evidenced by its ability to inhibit the oxidation of the fluorochrome dichlorofluorescin. 7. The results indicate a range of potential beneficial effects of quercetin with respect to the influence on carcinogen-metabolising enzymes, scavenging of reactive oxygen species and a lack of stress response in HepG2 cells.

Animals↗

PubMed1996

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Animals↗

PubMed1996

Effect of quercetin on the genotoxic potential of cisplatin.

The natural product flavonoid quercetin has been shown to sensitise cells to the cytotoxic potential of cisplatin. Both cisplatin and quercetin are genotoxicants. As quercetin is currently in clinical trial as a cytotoxicant-sensitising agent, we wanted to elucidate whether it affects the genotoxicity associated with cisplatin. The genotoxic potential of both agents alone and in combination was studied in Salmonella typhimurium strains TA 98, TA 100 and TA 102 and by assessment of unscheduled DNA synthesis (UDS) in rat hepatocytes. Furthermore, effects of quercetin on levels of cisplatin-DNA adducts were studied in hepatocytes by ELISA. Cisplatin was mutagenic in all 3 bacterial strains and quercetin in strain TA 98. The number of revertant Salmonella colonies observed with the combination did not differ significantly from that caused by the drugs on their own. In the UDS assay, cisplatin was genotoxic but quercetin was not. In combination, quercetin decreased the nuclear grain count caused by cisplatin, but quercetin did not alter the level of cisplatin-DNA adduct formation in hepatocytes. Our results suggest that the mutagenic potential of the combination cisplatin-quercetin, as judged by the bacterial short-term test, does not exceed that associated with the individual components. However, in hepatocytes, quercetin appears to inhibit repair of cisplatin-induced DNA damage. Therefore, in patients who are to be treated with a combination of cisplatin and quercetin, the risk of genotoxicity in normal tissues will have to be taken into consideration.

Animals↗

PubMed1996

Comparison of the effects of redox cycling and arylating quinones on hepatobiliary function and glutathione homeostasis in rat hepatocyte couplets.

Menadione (2-methyl-1,4-naphthoquinone, a redox cycling and arylating quinone; 5-100 microM) inhibited the canalicular vacuolar accumulation (CVA) of a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), in rat hepatocyte couplets. This was associated with depletion of reduced glutathione and accumulation of oxidized glutathione, the latter indicating that the concentrations of menadione used were able to induce oxidative stress. There was no associated cytotoxicity as indicated by ATP content. Treatment of couplets with the redox cycling quinone 2,3-dimethoxy-1,4-naphthoquinone (up to 100 microM) had relatively little effect on CVA, suggesting that the magnitude of reactive oxygen formation induced by this compound was insufficient to disrupt canalicular integrity. In comparison, the arylation of protein thiol groups by p-benzoquinone (up to 100 microM) proved to be more potent in inhibiting canalicular vacuolar accumulation. The predominant mechanism of menadione-induced inhibition of couplet hepatobiliary function is therefore more likely to involve the arylation of critical thiol groups (such as those in the F-actin cytoskeleton) rather than their oxidation. The oxidative effects of menadione could, however, potentiate the deleterious effects induced by arylation, such as by reduced glutathione depletion.

Adenosine Triphosphate↗