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Archives of Toxicology (v.78, #2)
Concentrations of the des-F(6)-quinolone garenoxacin in plasma and joint cartilage of immature rats by Michael Kastner; Ute Rahm; Irmela Baumann-Wilschke; Akintunde Bello; Ralf Stahlmann (pp. 61-67).
Garenoxacin is a des-F(6)-quinolone with a broad antibacterial spectrum, which has previously been shown to exhibit low chondrotoxicity in juvenile dogs compared with several other quinolones. A study was performed to determine whether the low chondrotoxicity observed in immature rats following garenoxacin treatment could be explained by poor penetration into cartilage tissue. Garenoxacin was orally administered to immature (4- to 5-week-old) Wistar rats as a single dose—or as doses given on 5 consecutive days—of 0 (vehicle), 200, 400, and 600 mg/kg (n=5 per dose level). Additional groups of rats were orally dosed with 600 mg/kg ofloxacin and ciprofloxacin. One knee joint of each animal (24 h after the last dose) was studied histologically after staining with Toluidine blue. The pharmacokinetics of garenoxacin in plasma (200, 400, and 600 mg/kg) and in knee joint cartilage (200 and 600 mg/kg) was assessed in separate groups of rats (n=55 per dose level). Concentrations of garenoxacin in plasma and cartilage were measured using an HPLC method. No signs of chondrotoxicity were observed in the immature rats treated with garenoxacin or ciprofloxacin for 5 days at the doses investigated in this study. However, ofloxacin was found to induce cartilage lesions that were typical of those seen for this quinolone. Systemic exposure to garenoxacin increased as a function of dose. Across dose and study day, mean garenoxacin plasma maximum concentration (C max) and area under the concentration–time curve (AUCtau) values were in the range 12–26 mg/l and 33–133 mg×h/l, respectively. Garenoxacin C max and AUC were similar on days 1 and 5, within each dose, indicating the absence of accumulation or reduction in the systemic exposure. Values determined for T max (0.25–1.0 h) and T 1/2 (3.8–6.4 h) of garenoxacin in plasma did not vary with dose or study day. Although peak garenoxacin concentrations in cartilage were between equal levels to and 2.5-fold of those found in plasma, the observed ratios were somewhat lower than those reported for other quinolones, e.g. ofloxacin or sparfloxacin. Since garenoxacin appeared to be well absorbed following oral administration and concentrations in cartilage tended to be higher than those in plasma, it is unlikely that the low chondrotoxicity in comparison with other quinolones is explained by differences in the pharmacokinetics of these compounds.
Keywords: Quinolones; Garenoxacin; Pharmacokinetics; Cartilage; Joint histology; Juvenile rat
The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin α-amanitin by Sri Gundala; Lisa D. Wells; Michael T. Milliano; Vanita Talkad; Bruce A. Luxon; Brent A. Neuschwander-Tetri (pp. 68-73).
Hepatotoxicity caused by the mushroom poison α-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of α-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for α-amanitin uptake, we tested the hypothesis that Na+-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular α-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K m and V max. A toxicologically relevant functional assay for α-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1β (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 µM α-amanitin for 6–10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without α-amanitin). These findings demonstrate that Ntcp may be an important mediator of α-amanitin uptake by the liver.
Keywords: HepG2 cells; Stable transfection; Interleukin-1 receptor antagonist; Green fluorescent protein
Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells by A. G. Cabado; F. Leira; M. R. Vieytes; J. M. Vieites; L. M. Botana (pp. 74-85).
Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.
Keywords: Apoptosis; Okadaic acid; Cytoskeleton; Mitochondria; Neuroblastoma cells
Hepatotoxin-induced hypercreatinaemia and hypercreatinuria: their relationship to one another, to liver damage and to weakened nutritional status by T. Andrew Clayton; John C. Lindon; Jeremy R. Everett; Claude Charuel; Gilles Hanton; Jean-Loic Le Net; Jean-Pierre Provost; Jeremy K. Nicholson (pp. 86-96).
Hypercreatinuria is a well-known feature of liver and testicular toxicity and we have recently proposed that hepatotoxin-induced hypercreatinuria would arise as a consequence of increased cysteine synthesis associated with the provision of protective substances (glutathione and/or taurine). Here a direct relationship between hepatotoxin-induced hypercreatinaemia and hypercreatinuria is shown and the possible relationships of hepatotoxin-induced hypercreatinaemia and hypercreatinuria to hepatic damage and to weakened nutritional status are examined. Male Sprague-Dawley rats were dosed with a variety of model hepatotoxins at two dose levels per toxin. Blood plasma samples taken at 24 h post-dosing and urine samples collected from 24–31 h post-dosing were analysed by 1H NMR spectroscopy. Both hypercreatinaemia and hypercreatinuria were found in rats dosed with allyl formate (75 mg/kg), chlorpromazine (30 and 60 mg/kg), α-naphthylisothiocyanate (ANIT, 100 mg/kg) and thioacetamide (200 mg/kg), whilst significant hypercreatinuria, but not hypercreatinaemia, was found after dosing with thioacetamide (50 mg/kg). Neither hypercreatinaemia nor hypercreatinuria were found after dosing with allyl formate (25 mg/kg), ethionine (300 and 1000 mg/kg) or ANIT (30 mg/kg). Reduced feeding is known to cause hypercreatinuria in rats and, of the four hepatotoxins that induced hypercreatinaemia and hypercreatinuria at the given time-points, two, chlorpromazine and ANIT, also affected nutritional status with ketosis being clearly identifiable from the plasma 1H NMR spectra. Thus, the creatine changes induced by ANIT and chlorpromazine are potentially attributable, in whole or in part, to reduced feeding rather than to liver effects alone and, consequently, the results were examined with and without inclusion of the ANIT and chlorpromazine data. With all of the data included, there were eight out of ten points of correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma alanine aminotransferase (ALT) activity. At the same time there were nine out of ten points of correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma aspartate aminotransferase (AST) activity. However, with the ANIT and chlorpromazine data excluded there was complete (six out of six points) correspondence between the incidence of hypercreatinaemia and/or hypercreatinuria and the incidence of increases in plasma AST and ALT in the remaining data. Likewise, with all of the data included, there was some apparent correlation (correlation coefficient, r=0.80) between the group mean levels of plasma AST and plasma creatine when expressed relative to the mean values for controls sampled at the same time-point. However, with the ANIT and chlorpromazine data excluded, that correlation coefficient was increased to 0.95. The findings of these studies suggest that the ANIT- and chlorpromazine-induced creatine changes may have been caused by reduced feeding rather than by liver toxicity. The allyl formate and thioacetamide data indicate that hepatocellular necrosis is accompanied by increases in plasma and urinary creatine, and suggest the possibility of a quantitative relationship between the increases in plasma AST and the increases in plasma creatine that are associated with hepatocellular necrosis. The ethionine and ANIT data suggest that fatty liver (steatosis) and cholestatic damage may not be associated with hypercreatinaemia and hypercreatinuria.
Keywords: Creatine; Hypercreatinaemia; Hypercreatinuria; Hepatotoxin; NMR
Lack of maternal dietary exposure effects of bisphenol A and nonylphenol during the critical period for brain sexual differentiation on the reproductive/endocrine systems in later life by Hironori Takagi; Makoto Shibutani; Naoya Masutomi; Chikako Uneyama; Noriyuki Takahashi; Kunitoshi Mitsumori; Masao Hirose (pp. 97-105).
Two potential endocrine-disrupting chemicals, bisphenol A (BPA) and nonylphenol (NP), were assessed for their long-lasting effects on endocrine/reproductive systems following transplacental and lactational exposure to rat offspring during a time-window that included the critical period for brain sexual differentiation. Each chemical was mixed with diet at concentrations of 60, 600 and 3000 ppm and was provided to maternal Sprague-Dawley rats from gestational day (GD) 15 to postnatal day (PND) 10. Ethinylestradiol (EE) at 0.5 ppm was used as an estrogenic reference drug. During pregnancy and lactation, including the exposure period, a soy-free rodent diet was provided to eliminate possible modification of the study results by plant-derived phytoestrogens. Effects on endocrine/reproductive systems were evaluated by examining the anogenital distance, organ weights before puberty, onset of puberty, estrous cyclicity, and organ weights and histopathology of adult endocrine organs (at 11 weeks of age), as well as the volume of the sexually dimorphic nucleus of preoptic area. Both NP and BPA, at high doses, caused decreases in maternal body weights and retardation of offspring growth, but neither affected any of the endocrine/reproductive endpoints of offspring, whereas EE induced irreversible changes in estrous cyclicity and histopathology of ovaries and uterus of adult females. The results indicated that maternal dietary exposure to NP or BPA at concentrations up to 3000 ppm from GD 15 through PND 10 do not exert any apparent adverse effects on the endocrine/reproductive systems of offspring.
Keywords: Bisphenol A; Nonylphenol; Maternal exposure effect; Dietary exposure; Endocrine/reproductive systems
Electrophilic tissue burden in male Sprague-Dawley rats following repeated exposure to binary mixtures of polycyclic aromatic hydrocarbons by Adela Tzekova; Suzanne Leroux; Claude Viau (pp. 106-113).
The aim of this study was to investigate the electrophilic tissue burden (ETB) formation, assessed as covalent binding of the ultimate carcinogen benzo(a)pyrene diolepoxide (BaPDE) with cellular proteins, in liver, lung and heart, as well as with haemoglobin (Hb) following repeated exposure to binary mixtures of benzo(a)pyrene (BaP) and pyrene (P). Male Sprague-Dawley rats were injected intraperitoneally, once daily for 10 consecutive days, with binary mixtures of BaP and P in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5 and with three dose levels of BaP (2, 6 and 20 mg/kg) for each scenario. ETB levels were measured as the ultimate analyte benzo(a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE-adducts with proteins. A high-performance liquid chromatography fluorescence technique was used to quantify the analyte. Similar ETB levels (within a factor of 4) were observed in all tissues studied at any given binary dose. However, the ETB generally tended to be somewhat higher in metabolically active tissues (i.e. liver and lung) than in metabolically non-active tissues (i.e. heart and Hb). Lack of influence of pyrene on ETB levels in all tissues was confirmed over the binary dose range examined. Linear BaP-dose-dependent ETB formation in all tissues (at P≤0.05) was observed. Linear regressions were found for all between-tissue relationships of ETB over the exposure doses, with best linear correlations obtained for ETB in heart versus Hb (R 2=0.709; P<0.0001) and ETB in lung versus Hb (R 2=0.507; P<0.0001). The results thus suggest that BaPDE–Hb adducts could serve as a surrogate of the ETB, namely in tissues that are potential targets for carcinogenicity such as lung. The results obtained in this study indicate the role of the ETB as a promising molecular biomarker of the potential cellular damage resulting from intracellular covalent binding in animal studies.
Keywords: Benzo(a)pyrene; Benzo(a)pyrene diolepoxide; Polycyclic aromatic hydrocarbon; Pyrene; Electrophilic tissue burden
DNA adduct formation and physiological effects from crude oil distillate and its derived base oil in isolated, perfused rat liver by Eszter Nagy; Ulf G. Norén; Magnus Zeisig; Lars-Gösta Ekström; Lennart Möller (pp. 114-121).
A distillate, D431, originating from Venezuelan heavy crude oil and a severely hydro-treated base oil, BO100, derived from this distillate, were tested for DNA adduct formation capacities and overall impact on liver functions. D431 had earlier showed DNA adduct formation in vitro but not in vivo in the rat. In this study, isolated rat liver perfusions were performed to elucidate whether the lack of DNA adducts in vivo was because of lack of uptake or metabolism. The oils were extracted with dimethyl sulfoxide and the extracts added to the perfusion system. Bile production, lactate metabolism and perfusate flow through the organ, which are parameters that reveal the condition of the liver, were continuously monitored. Samples of liver were collected once every hour during perfusion for DNA adduct analysis with 32P-HPLC. The results for the distillate D431 showed that the production of bile and metabolism of lactate decreased while DNA adduct formation increased with time. The DNA adduct pattern formed in the D431-treated livers was similar to that found earlier in in vitro studies performed on calf thymus DNA (CT-DNA). In the case of DNA adduct formation, virtually no difference with dose was seen, suggesting that perhaps a point of saturation of, for instance, enzymatic systems was reached. The results for base oil BO100 showed no significant difference regarding bile production, lactate metabolism and DNA adduct formation when compared with the control, indicating no toxic or genotoxic activity.
Keywords: DNA adducts; Liver; Crude oil distillate; 32P-HPLC; Rat