Right ventricular ejection fraction measured by multigated planar equilibrium radionuclide ventriculography is an independent prognostic factor in patients with ischemic heart disease.

BACKGROUND: The number of studies on the prognostic value of the right ventricular ejection fraction (RVEF) in patients with ischemic heart disease (IHD) is limited, whereas it is widely accepted that the left ventricular ejection fraction (LVEF) is a strong prognostic factor. We assessed whether RVEF measured by multigated planar equilibrium radionuclide ventriculography (RNV) is an independent prognostic factor in patients with IHD. METHODS AND RESULTS: We retrospectively identified 347 consecutive patients with IHD (mean age 71 ± 11 years; 18% women) who underwent multigated planar equilibrium RNV between 2004 and 2008 to determine the LVEF, which also provided the RVEF (mean 44.7% ± 11.0%). We categorized patients according to RVEF in ≥40% (n = 240) and <40% (n = 107). Patients were followed for a median of 826 days (range 3-2,400) for the occurrence of events [all-cause mortality (n = 60), cardiac mortality (n = 33), and cardiac hospitalization (n = 78)]. Cox regression analysis with significant univariate predictors [coronary artery revascularization (P = .003), diuretics (P = .03), and statins (P < .001)] showed that an RVEF <40% was associated with a 2.90 (1.68-5.00)-fold higher risk of all-cause death. Accordingly, a decreased RVEF was associated with a 2.15 (1.34-3.43)-fold increase in the risk of cardiac hospitalization and a 5.11(2.32-11.23)-fold risk of cardiac death. CONCLUSION: RVEF measured by multigated planar equilibrium RNV is an independent prognostic factor in patients with chronic IHD.

A kinase inhibitor screen reveals MEK1/2 as a novel therapeutic target to antagonize IGF1R-mediated antiestrogen resistance in ERα-positive luminal breast cancer.

Antiestrogen resistance of breast cancer has been related to enhanced growth factor receptor expression and activation. We have previously shown that ectopic expression and subsequent activation of the insulin-like growth factor-1 receptor (IGF1R) or the epidermal growth factor receptor (EGFR) in MCF7 or T47D breast cancer cells results in antiestrogen resistance. In order to identify novel therapeutic targets to prevent this antiestrogen resistance, we performed kinase inhibitor screens with 273 different inhibitors in MCF7 cells overexpressing IGF1R or EGFR. Kinase inhibitors that antagonized antiestrogen resistance but are not directly involved in IGF1R or EGFR signaling were prioritized for further analyses. Various ALK (anaplastic lymphoma receptor tyrosine kinase) inhibitors inhibited cell proliferation in IGF1R expressing cells under normal and antiestrogen resistance conditions by preventing IGF1R activation and subsequent downstream signaling; the ALK inhibitors did not affect EGFR signaling. On the other hand, MEK (mitogen-activated protein kinase kinase)1/2 inhibitors, including PD0325901, selumetinib, trametinib and TAK-733, selectively antagonized IGF1R signaling-mediated antiestrogen resistance but did not affect cell proliferation under normal growth conditions. RNAseq analysis revealed that MEK inhibitors PD0325901 and selumetinib drastically altered cell cycle progression and cell migration networks under IGF1R signaling-mediated antiestrogen resistance. In a group of 219 patients with metastasized ER + breast cancer, strong pMEK staining showed a significant correlation with no clinical benefit of first-line tamoxifen treatment. We propose a critical role for MEK activation in IGF1R signaling-mediated antiestrogen resistance and anticipate that dual-targeted therapy with a MEK inhibitor and antiestrogen could improve treatment outcome.

Transfer of uremic solutes across the human term placenta: An ex vivo study in the dual-side perfused cotyledon.

INTRODUCTION: An increasing number of women becomes pregnant while suffering from chronic kidney disease (CKD). As a result of decreased renal function, uremic solutes circulate at high levels in the maternal circulation. This study aimed to acquire more knowledge about the placental transfer of uremic solutes across the human placenta. METHODS: Placental transfer was studied in healthy term placentas, via the ex vivo dual-side human cotyledon perfusion technique (closed-closed set-up for both maternal and fetal circulations). Uremic solute concentrations in maternal and fetal perfusates were measured via LC-MS/MS over 180 min of perfusion. RESULTS: We found that the studied compounds demonstrated different degrees of placental transfer. Fetal-to-maternal perfusate ratios at t = 180 min were for anthranilic acid 1.00 ± 0.02, indole-3-acetic acid 0.47 ± 0.08, hippuric acid 0.36 ± 0.18, l-arabinitol 0.33 ± 0.04, indoxyl sulfate 0.33 ± 0.11, neopterin 0.28 ± 0.14 and kynurenic acid 0.13 ± 0.03. All uremic solutes studied also emerged in the perfusates when cotyledons were perfused in the absence of uremic solute concentrations added to the maternal reservoir. For kynurenin these concentrations were so high, it complicated the calculation of a transfer ratio for the exogenously administered compound. DISCUSSION: After 180 min of exposure the extent of placental transfer differs substantially for the solutes studied, reflecting different transfer rates. Future studies should investigate to what extent specific uremic solutes reach the fetal circulation in vivo and how they may interfere with organ function and development of the unborn child.

Application of lipid peroxidation and protein oxidation biomarkers for oxidative damage in mammalian cells. A comparison with two fluorescent probes.

We recently developed two biomarker sets for oxidative damage: one for determination of lipid peroxidation (LPO) degradation products; acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, malondialdehyde and acetone, by a gas chromatography-electron capture detection method, and the other for protein oxidation products such as o,o'-dityrosine, by an isotope dilution high performance liquid chromatography-tandem mass spectrometry method. In the present study, we explored the possibility to utilize these biomarkers for determining the oxidative damage in liver mammalian cells in vitro. Two different treatments were chosen for inducing oxidative stress in Chinese Hamster ovary cells: menadione and copper plus hydrogen peroxide (Cu2+/H2O2). Cells were incubated with the model compounds in the presence or absence of vitamin E and C, and cytotoxicity was evaluated by a nuclear-dye method. Results were compared to two fluorescent probes, H2DCF-DA and C11 -BODIPY581/591, which have been used for determining the formation of free radicals in the cells. From ten LPO degradation products, eight were increased significantly following incubation with menadione in cell lysate or incubation media. Menadione-induced oxidative stress was also confirmed by oxidation of fluorescent probes. However, no increased formation of protein oxidation products was observed. Vitamin E and C did not diminish the formation of LPO degradation products that were increased by menadione. Although Cu2+/H2O2 did not induce oxidation of fluorescent probes, it induced formation of six out of ten LPO degradation products. Vitamin E and C did not diminish the formation of LPO degradation products; vitamin C even substantially increased the formation of acetaldehyde and propanal, which is in line with its reported prooxidant action under certain conditions. Vitamin C also caused two-fold increase in Cu2+/H2O2-induced o,o'-dityrosine formation when applied simultaneously. In conclusion, our present results show that the LPO biomarker set can be used for evaluation of oxidant capacity and the toxic potential of various chemicals in an in vitro cell model. These biomarkers might even be more sensitive than measuring protein oxidation products or oxidation of fluorescent probes.

Effect of glutathione depletion on the hepatotoxicity and covalent binding rat liver macromolecules of N-hydroxy-2-acetylaminofluorene.

Glutathione plays an important role in the protection of the liver against several hepatotoxins. The hepatocarcinogen N-hydroxy-2-acetylaminofluorene is converted in the rat in vivo to reactive metabolites that bind covalently to cellular macromolecules. These metabolites may also react with glutathione, resulting in the formation of glutathione conjugates and in the detoxification of reactive metabolites. The role of glutathione in detoxification was investigated by depletion of glutathione in the rat in vivo with diethyl maleate. When rats were pretreated with diethyl maleate, 45 min before the administration of N-hydroxy-2-acetylaminofluorene, excretion of 2-acetylaminofluorene:glutathione conjugates in bile was decreased by 60% as compared to controls. However, total covalent binding to rat liver protein was not increased, and total binding to DNA was even decreased (p less than 0.1), apparently at the expense of the acetylated carcinogen-DNA adducts. Formation of deacetylated, 2-aminofluorene adducts to DNA was not affected by diethyl maleate. Pretreatment with diethyl maleate had no major effect on the acute hepatotoxic effects of N-hydroxy-2-acetylaminofluorene. The results indicate that glutathione does not play a vital role in the detoxification of reactive metabolites generated from the carcinogen N-hydroxy-2-acetylaminofluorene, since glutathione is not very effective in competing with macromolecules for trapping of reactive metabolites of N-hydroxy-2-acetylaminofluorene. Thus, 1 mM glutathione did not decrease the covalent binding of 2-acetylaminofluorene-N-sulfate (one of the main reactive metabolites that is formed in vivo) to DNA in vitro, while 10 mM glutathione decreased the covalent binding to RNA by only 20% and to DNA by only 40%.

Formation of cyclic 1,N2-propanodeoxyguanosine and thymidine adducts in the reaction of the mutagen 2-bromoacrolein with calf thymus DNA.

The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-3H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N2-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N2-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules. Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz 1H and 75.43 MHz 13C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2"-bromo-3"-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [3H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct. This indicates that reaction of 2BA with this nucleotide in DNA is a major reaction.

Blocking of in vitro DNA replication by deoxycytidine adducts of the mutagen and clastogen 2-bromoacrolein.

Calf thymus single-stranded DNA was modified with 2-bromoacrolein (2BA), a genotoxic metabolite of tris(2,3-dibromopropyl)phosphate. This DNA was used as a template for in vitro DNA replication by T7-polymerase and Klenow fragment of Escherichia coli DNA polymerase I. Increasing levels of 2BA modification led to decreased DNA synthesis as measured by [methyl-3H]dTTP incorporation. M13 mp19 single-stranded DNA template modified with 2BA was used to determine the sites of termination of DNA replication by T7 polymerase and Klenow fragment of Escherichia coli DNA polymerase I. It was found that DNA replication stopped frequently before and occasionally opposite deoxycytidine nucleotides. These results indicated that an as yet unidentified deoxycytidine:2BA adduct may have been formed in the reaction of 2BA with M13 DNA. To investigate if such adducts were formed, we reacted 2BA with deoxycytidine in vitro at pH 4.4, and putative deoxycytidine:2BA adducts were isolated by high-performance liquid chromatography. They were characterized by 1H and 13C nuclear magnetic resonance and with fast atom bombardment mass spectrometry as two diastereomeric 3-bromo-7-(beta-D-deoxyribofuranosyl)- 3,4-dihydro-2-hydroxy-(2H,7H)[1,6-a]pyrimidin-6-one adducts and a 3-bromo-7-(beta-deoxyribofuranosyl)-(4H,7H)-pyrimido[1,6-a]pyrimidin-6 -one adduct. Only the latter adduct, however, was formed in the reaction of 2BA with calf thymus single-stranded DNA in vitro. Tris(2,3-dibromopropyl)phosphate is clastogenic. Because clastogenicity may result from DNA adducts that block replication, the role of the presently identified deoxycytidine adducts of the reaction metabolite 2BA in the clastogenicity of tris(2,3-dibromopropyl)phosphate is discussed.

Biomarkers of free radical damage applications in experimental animals and in humans.

Free radical damage is an important factor in many pathological and toxicological processes. Despite extensive research efforts in biomarkers in recent years, yielding promising results in experimental animals, there is still a great need for additional research on the applicability of, especially non-invasive, biomarkers of free radical damage in humans. This review gives an overview of the applications in experimental and human situations of four main groups of products resulting from free radical damage, these include: lipid peroxidation products, isoprostanes, DNA-hydroxylation products and protein hydroxylation products.

Lack of p53 protein expression in preneoplastic rat hepatocytes in vitro after exposure to N-acetoxy-acetylaminofluorene, X-rays or a proteasome inhibitor.

Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.

Simulations of the estrogen receptor ligand-binding domain: affinity of natural ligands and xenoestrogens.

We have carried out molecular dynamics (MD) simulations and free energy calculations on the alpha-subtype of the human estrogen receptor ligand-binding domain (ERalpha LBD) complexed with a number of known agonists and putative xenoestrogens. Our dynamical simulations of ligand-receptor complexes underscore the highly structured nature of the complex and offer some interesting insights into the structure-activity relationship (SAR) for these ligands. With traditional thermodynamic integration (TI) calculations, we calculate relative binding free energies for three known agonists, in good agreement with experimental values. The sheer number of possible xenoestrogenic compounds makes an approach using traditional free energy calculations unfeasible. Instead, we have made use of a single-step perturbation methodology that allows the calculation of relative free energies for a large number of related polyaromatic hydrocarbons (PAHs) from a single simulation. Our results show good (maximum deviation 3.3 kJ mol(-1)) agreement with experimental data, suggesting the possibility of large-scale xenoestrogen screening in silico to obtain strongly estrogenic compounds for subsequent experimental testing.

Sex differences in sulfation and glucuronidation of phenol, 4-nitrophenol and N-hydroxy-2-acetylaminofluorene in the rat in vivo.

Sulfation and glucuronidation of phenol, 4-nitrophenol (4NP) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied in adult (60 days) male and female rats. Within 3 hours almost 50% of a dose of phenol was excreted in urine as phenyl sulfate; male rats excreted slightly more phenyl sulfate than females. This probably was due to a slower excretion of phenyl sulfate by the females. No sex difference in glucuronidation of phenol was found. Over a period of 24 hours male and female rats excreted almost 35% of a dose of 4NP as 4NP-sulfate in urine and almost 40% as 4NP-glucuronide. No differences in the excretion of 4NP-conjugates were found between sexes. However, almost twice as much of a dose of N-OH-AAF was excreted after 4 hours as the N-O-glucuronide in bile and urine in female than in males. On the other hand, females excreted less of the AAF-glutathione conjugates that are derived from the reaction of AAF-N-sulfate with glutathione in vivo [Meerman et al., Chem.-Biol. Interactions, 39, 149, 1982] in bile, than males. This indicates that sulfation of N-OH-AAF is less active in females than in males. Most likely, sulfation of the phenols is catalyzed by a different sulfotransferase than that of N-OH-AAF.

Sulphation of N-hydroxy-4-aminobiphenyl and N-hydroxy-4-acetylaminobiphenyl by human foetal and neonatal sulphotransferase.

Sulphation of the genotoxic compounds N-hydroxy-4-aminobiphenyl (N-OH-4ABP) and N-hydroxy-4-acetylaminobiphenyl (N-OH-4AABP) was determined in cytosolic preparations of human foetal, neonatal and adult liver and foetal and neonatal adrenal gland. Sulphotransferase (ST) activity capable of sulphating these compounds was present in foetal liver and adrenal gland by 14 weeks of gestation. Sulphation of N-OH-4ABP was higher in foetal and neonatal adrenal cytosol than was sulphation of N-OH-4AABP and in general, N-OH-4ABP ST activity was also greater than that towards 1-naphthol. In foetal and neonatal liver cytosol the sulphation of N-OH-4ABP was also higher than that of N-OH-4AABP (approximately 2-fold). In adult liver cytosols, however, N-OH-4AABP ST activity was higher than that for N-OH-4ABP and 1-naphthol sulphation. Aromatic hydroxylamines and hydroxamic acids are known to be converted by sulphotransferase into reactive, electrophilic compounds capable of reacting with DNA. Our data show that the human foetus and neonate have the capacity to sulphate these compounds and thus is able to produce the reactive mutagenic metabolites. Therefore, this class of genotoxic compounds may be bioactivated by humans during development--a time when they are most vulnerable to the effects of genotoxins.

Immobilization of solubilized UDP-glucuronosyltransferase from rat liver microsomes to Sepharose 4B.

A method for the covalent binding of rat liver UDP-glucuronosyltransferase to a cyanogen bromide-activated agarose matrix is described. The rat liver microsomal fraction was solubilized with 8 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 90% of the microsomal protein was solubilized. Some 50-60% of this protein became bound covalently to the activated agarose matrix. The immobilized UPD-glucuronosyltransferase remained completely active for 50 days when stored at 4 degrees in a 20% (v/v) glycerol buffer (pH 7.4). The immobilized enzyme has a temperature optimum around 37 degrees, and a broad pH optimum (pH 5-7.4). The enzyme displayed linear kinetics over a period of 1 hr; it conjugates a large variety of substrates.

Use of pentachlorophenol as long-term inhibitor of sulfation of phenols and hydroxamic acids in the rat in vivo.

Inhibition of sulfation of the phenolic compound harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole) by pentachlorophenol (PCP) was studied in the Wistar rat: PCP was administered in various ways to find a convenient method for long-term inhibition of sulfation. High doses of PCP or sodium pentachlorophenolate (NaPCP) in the diet (350 ppm) or NaPCP in the drinking water (1.4 mM) of Wistar rats for one week inhibited the sulfation of harmol by 30-45%. The plasma concentration of PCP in rats with NaPCP (1.4 mM) in their drinking water was highest (270 microM) in the period that the animals were kept in the dark and consumed food and water. This is explained by a rapid elimination: the elimination of PCP from plasma, after intravenous administration, showed a biphasic disappearance curve with half-lives of 2.17 and 7.24 hrs, respectively. This is much faster than in Sprague-Dawley rats. A log-linear correlation was found between the plasma concentration of pentachlorophenol and the inhibition of harmol sulfation. Although administration of NaPCP to rats in their drinking water inhibited the sulfation of harmol only by 45%, it inhibited the sulfation of the carcinogenic arylhydroxamic acid N-hydroxy-2-acetylaminofluorene by 70-75%.

Selective inhibition of sulfate conjugation in the rat: pharmacokinetics and characterization of the inhibitory effect of 2,6-dichloro-4-nitrophenol.

The pharmacokinetics of 2,6-dichloro-4-nitrophenol (DCNP) have been studied in the rat. Upon i.v. injection the plasma decay curve of DCNP showed a rapid distribution phase. After 30 min the plasma concentration reached a value that was constant for at least 90 min, indicating very slow elimination of DCNP. The volume of distribution was 88 ml/kg and a high degree of binding (over, 99%) of DCNP in vitro to bovine serum albumin was found. The concentration of DCNP in the liver was between 30 and 50% of the plasma values. While in vivo the effect of DCNP persisted for a long time, its action was readily reversible in the single-pass perfused rat liver. In vivo, the effect of the dose of DCNP on the inhibition of sulfation of the phenolic compound harmol was investigated. Upon the i.v. injection of 26 mumole DCNP/kg an instantaneous and complete inhibition of sulfation of harmol was found. Using this property of DCNP, the rate of sulfation of harmol in vivo was evaluated in relation to the dose and the time after injection of the substrate. Saturation of sulfation apparently occurred because the consumption of inorganic sulfate was extremely small.

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes.

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.

4-Methylthiobenzoic acid reduces cisplatin nephrotoxicity in rats without compromising anti-tumour activity.

Administration of 4-methylthiobenzoic acid (MTBA) (100 mg/kg) strongly reduced cisplatin nephrotoxicity (7.5 mg/kg, 20 min after MTBA) in rats as determined by histopathology and blood urea nitrogen. Anti-tumour activity against a colonic adenocarcinoma, CC 531, that was implanted in rats, was unaffected by MTBA pretreatment. Studies with isolated renal proximal tubular cells (PTC) demonstrated that preincubation of the cells with MTBA diminished cisplatin nephrotoxicity in vitro as it did in vivo. Preincubation of the PTC with probenecid completely abolished the protective effect of MTBA against cisplatin toxicity. These data indicate that MTBA is actively transported into the PTC. The mechanism of action of MTBA was investigated by NMR studies which showed that cisplatin and cis-diamminediaquaplatinum(II), its hydrolysis product, reacted with the methylthio-sulphur. We suggest that MTBA after selective accumulation in the kidney inactivates cisplatin intracellularly by nucleophilic attack of the methylthio-sulphur to the Pt-moiety. Since MTBA shows no acute toxicity in the rat, even if administered at very high doses, it may be useful to suppress the nephrotoxic side effects of cisplatin anti-tumour therapy.

Fasting increases the susceptibility of rat hepatocytes to the cytotoxic effects of N-hydroxy-acetylaminofluorene. Effects on mitochondrial respiration and membrane potential.

Isolated rat hepatocytes were incubated with the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Cells from fasted rats were much more susceptible to the cytotoxic effects of 1 mM N-OH-AAF than cells from fed rats: after approximately 90 min exposure the former were all dead but the latter still viable. Even after 240 min 25% of the "fed" cells were still viable. The loss of viability was preceded by a decrease in mitochondrial membrane potential (MMP) and inhibition of respiration; the mitochondrial respiration as measured in permeabilized cells appeared uncoupled. Addition of 15 mM fructose prevented cell death and the loss of MMP in cells both from fed and fasted rats to a large extent; however, uncoupling was not prevented. After incubation of hepatocytes from fasted rats with 1 mM [3H]N-OH-AAF for 120 min, 12 nmol [3H]N-OH-AAF became bound per mg cell protein. Addition of fructose decreased this to 7 nmol. In cells from fed animals 4 nmol [3H]N-OH-AAF became bound after 120 min, in this case fructose had no effect. Part of the protective effect of fructose might be explained by a decrease in intracellular ATP, which prevents the formation of reactive intermediates of N-OH-AAF resulting in a decrease of covalent binding, in addition, fructose protects via a yet to be determined mechanism.

Sulfation and glucuronidation as competing pathways in the metabolism of hydroxamic acids: the role of N,O-sulfonation in chemical carcinogenesis of aromatic amines.

Aromatic amines can be metabolized by N-acetylation and N-hydroxylation to hydroxamic acids; these subsequently are conjugated to form the N,O-sulfonate and N,O-glucuronide conjugates. The N,O-sulfonates are highly labile metabolites that generate reactive intermediates involved in the covalent binding of the parent compound to protein, RNA and DNA, as well as to low molecular compounds like glutathione. This paper discusses methods used to decrease sulfation in vivo, and thereby to enhance the formation of the more stable N,O-glucuronides from N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylamino-4'-fluorobiphenyl. Acetaminophen pretreatment decreases the sulfate availability, but results in many side effects that complicate the analysis of the results. An 8% casein diet reduces the sulfate availability in the rat to approximately 20% of control and thus offers an effective approach to decrease sulfation. The most effective selective inhibition of sulfation is by pentachlorophenol, which very strongly reduces N,O-sulfonation of both hydroxamic acids, and selectively inhibits the formation of DNA adducts that have retained the N-acetyl group. This inhibitor and the related 2,6-dichloro-4-nitrophenol can be employed to study the role of sulfation of hydroxamic acids in initiation and promotion of tumor formation by aromatic amines.

Metabolic activation routes of arylamines and their genotoxic effects.

Two different types of DNA adducts are formed from many aromatic amines by bioactivation: N-acetylated and nonacetylated, arylamine DNA adducts. It has become clear from experiments using N-acetyl-2-aminofluorene and 2-aminofluorene adducts to C8 of deoxyguanosine that these two types of adducts may have different effects on DNA structure and DNA replication. We have determined blocking of DNA replication by various other N-acetylarylamine and arylamine deoxyguanosine adducts. It was found that the N-acetyl group in general is required for blocking of DNA replication; the nature of the aromatic moiety seems to be of minor importance. Little information is available on the genotoxic effects of these adducts in mammalian cells in vivo. We have tried to get more insight in this by investigating the clastogenicity, the initiation of preneoplastic cells, and the promotional effects of various aromatic amines from which different ratios of N-acetylarylamine DNA adducts to arylamine DNA adducts are formed in the rat liver. Our results show that formation of N-acetylarylamine adducts to C8 of deoxyguanosine in the liver is correlated with clastogenicity and hepatic promoting effect. Initiation capacities, however, seem to be correlated with formation of nonacetylated, arylamine adducts. Mechanisms by which formation of N-acetylarylamine DNA adducts may generate a promoting effect in the liver are discussed.