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Archives of Toxicology (v.79, #10)
Enzyme kinetics of zearalenone biotransformation: pH and cofactor effects by H. Malekinejad; R. F. Maas-Bakker; J. Fink-Gremmels (pp. 547-553).
The aim of the present study was to investigate the hepatic biotransformation of the mycotoxin zearalenone (ZEA) in vitro using subcellular fractions of pig livers. The dependencies of the enzymatic reactions involved on the enzyme velocity, on the cofactor and on pH were analysed in both the microsomal fraction and the post-mitochondrial cell fraction. Finally, the inhibitory effects of various endogenous substrates on the enzymes involved (3α- and 3β-hydroxysteroid dehydrogenase) were examined. Significant differences were observed between the individual subcellular fractions in terms of prevailing metabolites and absolute amounts of the metabolites produced. Moreover, this study also demonstrated that the reactions for both subcellular fractions of porcine liver are dependent on the cofactor, as α-zearalenol (α-ZOL) formation increased in the presence of NADPH, whereas β-zearalenol (β-ZOL) production only increased in the presence of NADH (P<0.001). The optimal pH for α-ZOL production was pH 5.6 and that for β-ZOL formation pH 7.4. Subsequent inhibition studies showed significant inhibitory effects for 5α-androstanedione>androstanedione>pregnenolone on α-ZOL formation, whereas β-ZOL production was only inhibited by pregnenolone. Finally, the contributions of 3α- and 3β-hydroxysteroid dehydrogenase during the bioconversion of ZEA are discussed in the context of these experiments.
Keywords: Zearalenone; α-Zearalenol; β-Zearalenol; Hepatic biotransformation; Cofactors; Hydroxysteroid dehydrogenase
The uridine diphosphate glucuronosyltransferases: quantitative structure–activity relationships for hydroxyl polychlorinated biphenyl substrates by Degao Wang (pp. 554-560).
Quantitative structure–activity relationships (QSARs), which relate the glucuronidation of hydroxyl polychlorinated biphenyls (OH-PCBs)—catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs)—to their physicochemical properties and molecular structural parameters, can be used to predict the rate constants and interpret the mechanism of glucuronidation. In this study, QSARs have been developed that use 23 semi-empirical calculated quantum chemical descriptors to predict the logarithms of the constants 1/Km and Vmax, related to enzyme kinetics. A partial least squares regression method was used to select the optimal set of descriptors to minimize the multicollinearity between the descriptors, as well as to maximize the cross-validated coefficient (Q2 cum) values. The key descriptors affecting log(1/Km) were Elumo− Ehomo (the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital) and qC− (the largest negative net atomic charge on a carbon atom), while the key descriptors affecting logVmax were the polarizability α, the Connolly solvent-excluded volume (CSEV), and logP (the logarithm of the partition coefficient for octanol/water). From the results obtained it can be concluded that hydrophobic and electronic aspects of OH-PCBs are important in the glucuronidation of OH-PCBs.
Keywords: Hydroxyl PCB; QSAR; UGT; Glucuronidation; PLS
Toxicant-induced ER-stress and caspase activation in the olfactory mucosa by Anna Franzén; Eva B. Brittebo (pp. 561-570).
The potent olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) induces rapid cell death and long-term metaplastic changes in the olfactory regions of rodents. The damage is related to a tissue-specific and extensive cytochrome P450 (CYP)-mediated metabolic activation of the compound to reactive intermediates. The aim of the present study was to examine the early, cell-specific changes leading to cell death in the olfactory mucosa of mice exposed to 2,6-diClPh-MeSO2. We have examined the expression of the ER-specific stress protein GRP78, the presence of secretory glycoproteins, and the cellular activation of the initiator caspase 12 and the downstream effector caspase 3. 2,6-DiClPh-MeSO2 induced rapid and cell-specific expression of GRP78, and activation of caspases 12 and 3 in the Bowman’s glands. No similar early onset changes in the neuroepithelium were observed. Based on these results, we propose that extensive lesions are initiated in the Bowman’s glands and that the metabolic activation of 2,6-diClPh-MeSO2 elicits ER-stress response and subsequent apoptotic signaling at this site. Since most of the Bowman’s glands had oncotic morphology, the results suggest that the terminal phase of apoptosis was blocked and that these glands finally succumb to other routes of cell death.
Keywords: Olfactory mucosa; Stress protein; Caspase 3; Caspase 12; Chlorinated benzene
Effect of methanol on endogenous and exogenous carnitine levels in rat plasma by Zofia Olszowy; Ewa Czech; Joanna Nowicka (pp. 571-581).
The effect of methanol on the levels of endogenous carnitine and its derivatives was studied in male Sprague-Dawley rats aged three months. In addition, the effect of L-carnitine supplementation on metabolic disturbances caused by methanol intoxication was studied. The rats were randomized into six groups, including two control groups. Methanol was given at 1/4 LD50 and 1/2 LD50/kg b.w. (or water in control) through an intragastric tube, and L-carnitine (or 0.9% NaCl in the control) was injected intraperitoneally. The levels of plasma L-carnitine and its derivatives were measured at selected time points for four days. Following methanol administration, the rats exhibited dose-dependent increases in L-carnitine levels and altered ratios of L-carnitine and its derivatives. L-carnitine supplementation accelerated the normalization of metabolic disturbances, as indicated by the acylcarnitine to free carnitine ratio (AC/FC). The protective effect of L-carnitine is supported by the fact that 100% of the methanol-treated rats supplemented with carnitine survived, while 8/60 rats and 27/101 rats died at methanol doses of 1/4 LD50 and 1/2 LD50, respectively, in groups without L-carnitine supplementation.
Keywords: L-Carnitine; Methanol; Rats
Effect of okadaic acid on integrins and structural proteins in BE(2)-M17 cells by Francisco Santaclara; Jorge Lago; Juan M. Vieites; Ana G. Cabado (pp. 582-586).
Okadaic acid (OA), an algal toxin, is known to induce Diarrhetic Shellfish Poisoning and apoptosis in a variety of cell lines. One of the main targets of OA is the actin cytoskeleton which can be modulated by integrins and other structural proteins. In this paper we studied the role of these proteins and skeletal structures on OA-induced apoptosis in neuroblastoma cells. Results show that β1 integrin and vinculin are down-regulated when cells were exposed to OA. We observed an interaction between talin and β1 integrin that is impaired in OA treated cells.
Identification of novel targets of cephaloridine in rabbit renal proximal tubules synthesizing glutamine from alanine by Jérôme Guitton; Agnès Conjard; Assaad Eid; Mireille Martin; Michelle Boghossian; Hélène Delage; Gabriel Baverel; Bernard Ferrier (pp. 587-594).
Cephaloridine, which accumulates in the renal proximal tubule, is a model compound used for studying the toxicity of antibiotics towards this nephron segment. Several studies have demonstrated that cephaloridine alters renal intermediary and energy metabolism, but the mechanism by which this compound interferes with renal metabolic pathways remains incompletely understood. In an attempt to improve our knowledge in this field, we have studied the influence of cephaloridine on the synthesis of glutamine, which represents a key metabolic process involving several important enzymatic steps in the rabbit kidney. For this, suspensions of rabbit renal proximal tubules were incubated for 90 and 180 min in the presence of 5 mM alanine, an important glutamine precursor, both in the absence and the presence of 10 mM cephaloridine. Glutamate accumulation and glutamine synthesis were found to be inhibited by cephaloridine after 90 and 180 min of incubation, and cephaloridine accumulation in the renal proximal cells occurred in a time-dependent manner. The renal proximal tubule activities of alanine aminotransferase and glutamate dehydrogenase, which initiates alanine removal and releases the ammonia needed for glutamine synthesis, respectively, were inhibited to a significant degree and in a concentration-dependent manner by cephaloridine concentrations in the range found to accumulate in the renal proximal cells. Citrate synthase and glutamine synthetase activities were also inhibited by cephaloridine, but to a much lesser extent. The above enzymatic activities were not found to be inhibited when they were measured after successive dilutions of renal proximal tubules incubated for 180 min in the presence of 5 mM alanine and 10 mM cephaloridine. When microdissected segments (S1–S3) of rabbit renal proximal tubules were incubated for 180 min with 5 mM alanine with and without 5 and 10 mM cephaloridine, glutamate accumulation and glutamine synthesis were also inhibited in the three renal proximal segments studied; the latter cephaloridine-induced inhibitions observed were concentration-dependent except for glutamine in the S3 segment. These results are consistent with the view that cephaloridine accumulates and is toxic along the entire rabbit renal proximal tubule. They also demonstrate that cephaloridine interferes in a concentration-dependent and reversible manner mainly with alanine aminotransferase and glutamate dehydrogenase, which are therefore newly-identified targets of the toxic effects of cephaloridine in the rabbit renal proximal tubule.
Keywords: Cephaloridine; Nephrotoxicity; Renal proximal tubule; Glutamine synthesis; Alanine aminotransferase; Glutamate dehydrogenase
Pulmonary exposure to diesel exhaust particles induces airway inflammation and cytokine expression in NC/Nga mice by Ken-ichiro Inoue; Hirohisa Takano; Rie Yanagisawa; Takamichi Ichinose; Akinori Shimada; Toshikazu Yoshikawa (pp. 595-599).
Although several studies have reported that diesel exhaust particles (DEP) affect cardiorespiratory health in animals and humans, the effect of DEP on animal models with spontaneous allergic disorders has been far less intensively studied. The Nc/Nga mouse is known to be a typical animal model for human atopic dermatitis (AD). In the present study, we investigated the effects of repeated pulmonary exposure to DEP on airway inflammation and cytokine expression in NC/Nga mice. The animals were randomized into two experimental groups that received vehicle or DEP by intratracheal instillation weekly for six weeks. Cellular profiles of bronchoalveolar lavage (BAL) fluid and expressions of cytokines and chemokines in both the BAL fluid and lung tissues were evaluated 24 h after the last instillation. The DEP challenge produced an increase in the numbers of total cells, neutrophils, and mononuclear cells in BAL fluid as compared to the vehicle challenge (P<0.01). DEP exposure significantly induced the lung expressions of interleukin (IL)-4, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-1α when compared to the vehicle challenge. These results indicate that intratracheal exposure to DEP induces the recruitment of inflammatory cells, at least partially, through the local expression of IL-4 and chemokines in NC/Nga mice.
Keywords: NC/Nga mice; DEP; Cytokine; Chemokine
Expression of lung resistance-related protein, LRP, and multidrug resistance-related protein, MRP1, in normal human lung cells in long-term cultures by Thomas Lehmann; Abdel-Rahman Wageeh Torky; Ekkehard Stehfest; Stefan Hofmann; Heidi Foth (pp. 600-609).
Transport processes form part of the body’s defense mechanism, and they determine the intracellular levels of many endogenous and exogenous compounds. The multidrug resistance-related protein MRP1 and the lung resistance-related protein LRP are associated with drug resistance against chemotherapeutics; they protect cells against toxic compounds. There is much experimental evidence to suggest that both of these transporter proteins serve important physiological functions. The expression of LRP and MRP1 was studied in normal human bronchial epithelial cells (NHBEC) and peripheral lung cells (PLC) obtained from explant cultures from morphologically-normal human lung tissue taken from patients with lung cancer. LRP (mRNA and protein) was detected in the cells of the bronchi as well as the peripheral lung with low (a factor of 2.6) inter-individual variation in the first generation. No significant alterations were noted for LRP within three-to-four generations in the same patient. LRP expression was not substantially different between cultures from different topographic regions of the human lung. MRP1 protein and MRP1 mRNA could also be detected in all of the NHBEC and PLC cultures studied, but with substantially higher (a factor of 7.7) intra-individual variation in the first generation than for LRP. MRP expression was the same for bronchial cells and PLC when the material was obtained from both sites. The level of mRNA for MRP1 was, in general, less stable than that for LRP. In multigeneration explant cultures, the levels of LRP mRNA and protein and MRP1 protein did not fluctuate greatly, but the level of MRP1 mRNA dropped to about 25% of the reference value within four generations (after about 8–10 weeks of culture). In one case, NHBEC subpassages were followed over a period of 20 weeks. In this system MRP mRNA levels increased by more than threefold, while levels of MRP1 protein and LRP mRNA and protein were expressed at almost constant rates.
Effect of cytochrome P450 inhibitors and anticonvulsants on the acute toxicity of acrylonitrile by Frederick W. Benz; Donald E. Nerland (pp. 610-614).
Some of the more striking expressions of toxicity are the tremors and seizures observed approximately 100 min after exposure of rats to an acutely toxic dose of acrylonitrile (AN). These early events are followed by a second wave of severe clonic convulsions that occur just prior to death at about 3–4 h. For AN, at least two chemical entities could produce these toxic effects, namely the parent AN molecule, the metabolically-released cyanide, or both. Which of these two agents is responsible for each of the symptoms of acute intoxication is not known. To help dissect the toxicity, it was anticipated that an effective inhibitor of the oxidative metabolism of AN to cyanide could help us to understand which toxic symptoms might be associated with each agent. Three inhibitors of oxidative metabolism were tested, namely SKF-525A, 1-benzylimidazole and metyrapone and one alternative substrate, ethanol. As compared to SKF-525A and metyrapone, both 1-benzylimidazole and ethanol were highly effective in reducing blood cyanide levels to insignificant levels in rats treated with an LD90 dose of AN. In addition, both agents abolished the early seizure activity, suggesting that this first phase of seizures is due to cyanide and not the parent molecule. 1-Benzylimidazole did not prevent the severe clonic convulsive phase preceding death, suggesting that these terminal convulsions are due to the toxic effects of the parent AN molecule. The CNS depressant ethanol was only partially effective in attenuating the terminal convulsions. None of these agents affected the incidence of AN-induced mortality, clearly establishing that, even in the absence of cyanide, the parent AN molecule is acutely toxic. The partial effectiveness of ethanol suggested that anticonvulsants might be of benefit. Both phenobarbital and phenytoin protected rats from both the early and terminal convulsions, while valproic acid was ineffective. These effects were not related to a reduction in blood cyanide levels but rather due to their inherent anticonvulsant activity.
Keywords: Acrylonitrile; Cyanide; Cytochrome P450; Enzyme inhibitors; Anticonvulsants