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Archives of Toxicology (v.77, #4)

Simultaneous analysis of naphthols, phenanthrols, and 1-hydroxypyrene in urine as biomarkers of polycyclic aromatic hydrocarbon exposure: intraindividual variance in the urinary metabolite excretion profiles caused by intervention with β-naphthoflavone induction in the rat by Eivor Elovaara; Virpi Väänänen; Jouni Mikkola (pp. 183-193).

Two fluorimetric HPLC methods are described for the quantification of naphthols, phenanthrols and 1-hydroxypyrene (1-OHP) in urine specimens obtained from male Wistar rats exposed to naphthalene, phenanthrene and pyrene. The polycyclic aromatic hydrocarbons (PAHs) were given intraperitoneally, either alone (1.0 mmol/kg body weight) or as an equimolar mixture (0.33 mmol/kg), using the same dosages for repeated treatments on week 1 and week 2. Between these treatments, PAH-metabolizing activities encoded by aryl hydrocarbon (Ah) receptor-controlled genes were induced in the rats with β-naphthoflavone (βNF). Chromatographic separation of five phenanthrols (1-, 2-, 3-, 4-, and 9-isomers) was accomplished using two different RP C-18 columns. Despite selective detection (programmable wavelengths), the quantification limits in the urine ranged widely: 1-OHP (0.18 µg/l) d-glucuronide served as measures for the completeness of enzymatic hydrolysis. Characteristic differences observed in the urinary disposition of naphthalene, phenanthrene, and pyrene are described, as well as important factors (dose, metabolic capacity, relative urinary output) associated with biomarker validation. This intervention study clarifies intraindividual variation in PAH metabolism and provides useful information for the development of new methods applicable in the biomonitoring of PAH exposure in humans.

Keywords: Monohydroxy polycyclic aromatic hydrocarbons Hydroxyphenanthrene isomers HPLC Fluorescence detection Urine Biomarkers

Cholestasis induced by chronic treatment with α-naphthyl-isothiocyanate (ANIT) affects rat renal mitochondrial bioenergetics by F. M. Ferreira; P. J. Oliveira; A. P. Rolo; M. S. Santos; A. J. Moreno; M. F. da Cunha; R. Seiça; C. M. Palmeira (pp. 194-200).

Chronic cholestasis is characteristic of many human liver diseases. Renal injury has been often associated with this type of disease. The aim of this study was to evaluate the effect of cholestasis on kidney mitochondrial bioenergetics following in vivo chronic administration of α-naphthyl-isothiocyanate (ANIT), a known cholestatic agent. Serum markers of renal injury, kidney morphology and endogenous adenine nucleotides were measured in ANIT-treated rats (80 mg/kg per week s.c. for 16 weeks). Changes in membrane potential and mitochondrial respiration as well as alterations in mitochondrial calcium homeostasis were monitored. Cholestatic animals shown no alterations in renal morphology when compared with control. Additionally, following chronic ANIT administration, no significant alterations in mitochondrial respiratory function have been shown. The phosphorylation capacity of cholestatic kidney mitochondria was enhanced. Associated with these parameters, mitochondria from treated animals exhibited a decreased susceptibility to disruption of mitochondrial calcium homeostasis, due to permeability transition induction. These data suggest that, despite being submitted to chronic treatment with ANIT, kidney mitochondria from cholestasis-induced rats present some defense mechanisms to circumvent this aggression. They show improved phosphorylative capacity and, moreover, a decreased susceptibility to mitochondrial permeability transition induction, probably due to adaptative mechanisms of calcium transport.

Keywords: Kidney mitochondria Bile acids Cholestasis Membrane potential Respiratory activity Permeability transition pore

AM404 and VDM 11 non-specifically inhibit C6 glioma cell proliferation at concentrations used to block the cellular accumulation of the endocannabinoid anandamide by Kent-Olov Jonsson; Anna Andersson; Stig O. P. Jacobsson; Séverine Vandevoorde; Didier M. Lambert; Christopher J. Fowler (pp. 201-207).

AM404 [N-(4-hydroxyphenyl)arachidonylamide] and VDM 11 [(5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] are commonly used to prevent the cellular accumulation of the endocannabinoid anandamide, and thereby to potentiate its actions. However, it has been reported that AM404 can produce an influx of calcium into cells, which might be expected to have deleterious effects on cell proliferation. In the present study, AM404 and VDM 11 were found to reduce C6 glioma cell proliferation with IC₅₀ values of 4.9 and 2.7 µM, respectively. The inhibition of cell proliferation following a 96-h exposure was not accompanied by dramatic caspase activation, and was not prevented by either a combination of cannabinoid and vanilloid receptor antagonists, or by the antioxidant α-tocopherol, suggestive of a non-specific mode of action. Similar results were seen with palmitoylisopropylamide, although this compound only produced significant inhibition of cell proliferation at 30 µM concentrations. AM404 (1 µM), VDM 11 (1 µM) and palmitoylisopropylamide (3–30 µM), i.e. concentrations producing relatively modest effects on cell proliferation per se, reduced the vanilloid receptor-mediated antiproliferative effects of anandamide, as would be expected for compounds preventing the cellular accumulation of anandamide (and thereby access to its binding site on the vanilloid receptor). It is concluded that concentrations of AM404 and VDM 11 that are generally used to reduce the cellular accumulation of anandamide have deleterious effects upon cell proliferation, and that lower concentrations of these compounds may be more appropriate to use in vitro.

Keywords: Anandamide Uptake inhibitors Cannabinoid receptors Vanilloid receptors Cell proliferation Glioma cells

An hypothesis for a mechanism underlying hepatotoxin-induced hypercreatinuria 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. 208-217).

As part of a wider metabonomic investigation into the early detection and discrimination of site-specific hepatotoxicity, male Sprague-Dawley rats were dosed with the model hepatotoxins allyl formate, ethionine and α-naphthylisothiocyanate (ANIT). Urine samples collected pre- and post-dose were examined by ¹H nuclear magnetic resonance (NMR) spectroscopy and the toxin-induced changes in urinary taurine and creatine excretion were quantified. Hypertaurinuria and hypercreatinuria were observed following allyl formate dosing, hypertaurinuria with no change in creatine excretion was observed after ethionine dosing, and hypotaurinuria and hypercreatinuria were observed after ANIT dosing. These changes are indicative of different effects on liver and it has been previously suggested that some hepatotoxin-induced changes in urinary taurine excretion may be due to altered hepatic cysteine utilisation. A related hypothesis is now presented that would explain the selective hypercreatinuria in terms of increased cysteine synthesis.

Keywords: Allyl formate Ethionine α-Naphthylisothiocyanate (ANIT) Creatine Taurine Cysteine Liver Rat

Effects of dexfenfluramine on aristolochic acid nephrotoxicity in a rat model for Chinese-herb nephropathy by Frédéric Debelle; Joëlle Nortier; Volker M. Arlt; Eric De Prez; Anne Vienne; Isabelle Salmon; David H. Phillips; Monique Deschodt-Lanckman; Jean-Louis Vanherweghem (pp. 218-226).

Chinese-herb nephropathy (CHN) is a progressive renal interstitial fibrosis initially reported after concomitant intake of an anorexigen, (dex)fenfluramine, and a Chinese herb (Aristolochia fangchi) containing nephrotoxic and carcinogenic aristolochic acid (AA). We thus tested the possible enhancing effect of the active enantiomer dexfenfluramine (DXF) on AA nephrotoxicity in a rat model for CHN. Groups of 12 salt-depleted male Wistar rats received daily subcutaneous injections of 7 mg/kg body weight DXF (DXF group), 7 mg/kg body weight AA (AA group), a combination of the same doses of AA and DXF (AA+DXF group), or vehicle (control group) for up to 35 days. Six animals per group were killed on day 10 and the remaining six on day 35. Renal function was evaluated by determining serum creatinine and urinary leucine aminopeptidase activity. Histological evaluation of kidney samples was performed and tubulointerstitial injuries were semiquantified. The DXF group did not differ from controls for any parameter. Similarly elevated serum creatinine levels, decreased leucine aminopeptidase enzymuria, and renal lesions were observed in the AA and the AA+DXF groups after both 10 and 35 days. The formation of specific AA–DNA adducts in liver and renal tissue samples was assessed by the ³²P-postlabelling method. Specific AA–DNA adduct levels were significantly increased in kidney tissues from AA+DXF rats compared with AA rats. These functional and histological data suggest that DXF does not enhance AA nephrotoxicity in a rat model for CHN. Further investigations are needed to clarify the mechanism by which DXF may enhance AA–DNA adduct formation.

Keywords: Aristolochic acid Dexfenfluramine Chinese-herb nephropathy Renal fibrosis DNA adducts

Toxic effects of profenofos on tissue acetylcholinesterase and gill morphology in a euryhaline fish, Oreochromis mossambicus by J. Venkateswara Rao; D. Shilpanjali; P. Kavitha; S. S. Madhavendra (pp. 227-232).

Acute and sub-acute studies of profenofos [O-(4-bromo-2-chlorophenyl)-O-ethyl-S-propyl phosphorothioate] on fish, Oreochromis (Tilapia) mossambicus, were carried out to assess the toxicity in relation to behaviour, morphology, and interactions with the targeted enzyme acetylcholinesterase (AChE, EC 3.1.1.7). Profenofos can be rated as highly toxic to O. mossambicus, with a median lethal concentration (LC₅₀) of 0.272±0.0177 mg/l. The inhibitory and recovery pattern of brain and gill AChE was studied in vivo after exposure to a single LC₅₀ and multiple exposures to sub-lethal concentrations (0.108 mg/l) for 28 days, respectively. The LC₅₀-exposed fish exhibited 90% inhibition of AChE activity in brain and gill in 24 h, and completely recovered within 23 days. Electron microscopy studies revealed an abnormal gill morphology, with distinct breakages in gill arches and rakers, along with deep lesions and erosions in the epithelium. Prolonged exposure at 0.108 mg/l also had similar effects, such as gill damage and AChE inhibition. The in vitro AChE study indicated that profenofos is neurotoxic and that it alters the apparent K _m values widely in a concentration-dependent manner, resulting in a competitive type of inhibition. Based on the K _i values, the sensitivity of AChE in brain was greater than that in gill tissue, at 2.38×10^-5 and 4.62×10^-5 M, respectively. The bioaccumulation values in head, body and viscera were estimated at regular intervals by gas chromatography method. The results indicated that the accumulation of profenofos was the highest in viscera followed by head and body, with depuration rates of 6.14, 0.16 and 0.12 µg/h, respectively.

Keywords: Oreochromis mossambicus (Tilapia mossambica) Profenofos Acetylcholinesterase Gill Bioaccumulation

Characterization of the toxicity, mutagenicity, and carcinogenicity of methacrylonitrile in F344 Rats and B6C3F1 mice by Abraham Nyska; Burhan I. Ghanayem (pp. 233-242).

Methacrylonitrile is an unsaturated aliphatic nitrile. It is widely used in the preparation of homopolymers and copolymers, elastomers, and plastics, and as a chemical intermediate in the preparation of acids, amides, amines, esters, and other nitriles. Methacrylonitrile was nominated for study by the National Cancer Institute (USA) because of the potential for human exposure, structural similarity to the known carcinogen acrylonitrile, and a lack of toxicity and carcinogenicity data. Doses selected for the 2-year study were based on the results of the 13-week gavage studies. Groups of 50 male and 50 female animals were exposed by gavage to 0, 3, 10, or 30 mg/kg in F344 rats, and 0, 1.5, 3 or 6 mg/kg in B6C3F1 mice, 5 days per week for 2 years. Urinary excretion of N-acetyl-S-(2-cyanopropyl)-l-cysteine (NACPC) and N-acetyl-S-(2-hydroxypropyl)-l-cysteine (NAHPC) were measured as markers of exposure at various time points after methacrylonitrile administration, and demonstrated that exposure of animals to methacrylonitrile occurred as intended. Urinary excretion of NACPC and NAHPC increased in rats and mice in a dose-dependent manner. In contrast to observations in rats, the ratios of NACPC/creatinine were generally higher in female than in male mice. Further, the ratios of NAHPC/creatinine in rats were significantly greater at all time points and all doses than the corresponding ratios of NACPC/creatinine in male and female mice. In both rats and mice, survival was not affected by treatment. In rats, mean body weights of the 30 mg/kg groups were less than those of the vehicle controls after weeks 21 and 37 for males and females, respectively. No treatment-related effect on body weight was seen in mice. There were no neoplasms (in either species) or non-neoplastic lesions (mice only) that were attributed to methacrylonitrile administration. In rats, the incidences of olfactory epithelial atrophy and metaplasia of the nose were significantly greater in 30 mg/kg males and females than those in the vehicle controls. Increased incidences of cytoplasmic vacuolation occurred in the liver of males and females. Testing methacrylonitrile in a battery of short-term in vitro and in vivo tests showed no evidence of genotoxicity. In conclusion, under the conditions of these 2-year gavage studies, there was no evidence of a carcinogenic activity of methacrylonitrile in male or female F344/N rats or B6C3F1 mice. Methacrylonitrile-related non-neoplastic lesions were seen in the nose and liver of rats.

Keywords: Methacrylonitrile Carcinogenicity F344/N rat B6C3F1 mouse Olfactory epithelium Atrophy

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Archives of Toxicology (v.77, #4)