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Archives of Toxicology (v.77, #10)
Constitutive and inducible levels of CYP1A1 and CYP1A2 in rat cerebral cortex and cerebellum by Michael M. Iba; Amijoy Storch; Anima Ghosal; Susan Bennett; Kenneth R. Reuhl; Herbert E. Lowndes (pp. 547-554).
We examined the constitutive and inducible levels of microsomal cytochromes P450 1A1 and 1A2 (CYP1A) in rat cerebral cortex and cerebellum at the level of proteins by western blot analysis, and by catalytic activities via ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD). In the cerebral cortex, cytochrome P450 1A1 (CYP1A1) protein was more abundant than cytochrome P450 1A2 (CYP1A2) protein. Treatment with β-naphthoflavone (β-NF) caused a slight decrease in the level of the former but induced the latter 5.8-fold. In the cerebellum, in contrast to the cerebral cortex, CYP1A1 protein was less abundant than CYP1A2 protein in untreated rats, and while β-NF treatment caused a 3.3-fold induction of CYP1A1 protein, it resulted in a 10-fold decrease in CYP1A2 protein. The CYP1A-preferential activity EROD was 2.3-fold higher in the cerebellum than in the cerebral cortex, and was induced 1.5-fold and 1.9-fold in the cerebellum and cerebral cortex, respectively, by β-NF treatment. The CYP1A2-preferential activity MROD was 3-fold higher in the cerebellum than in the cerebral cortex, and was repressed 2.2-fold in the cerebellum but induced 3.7-fold in the cerebral cortex following β-NF treatment. The results show that CYP1A1 and CYP1A2 proteins and catalytic activities are constitutively expressed in brain but are differentially inducible in the rat cerebral cortex and cerebellum.
Keywords: Cytochrome P450 1A1 (CYP1A1); Cytochrome P450 1A2 (CYP1A2); Brain; Cerebral cortex; Cerebellum; β-Naphthoflavone; Ethoxyresorufin O-deethylase (EROD); Methoxyresorufin O-demethylase (MROD)
Inter-species comparisons of hepatic cytochrome P450 enzyme levels in male ruminants by Miroslav Machala; Pavel Souček; Jiří Neča; Robert Ulrich; Jiří Lamka; Barbora Szotáková; Lenka Skálová (pp. 555-560).
Our current knowledge about the biotransformation enzymes in wild ruminants is limited. The present study aimed to compare basic levels and specific activities of cytochrome P450 isoforms (CYP1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A) in males of red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus) and mouflon (Ovis musimon). The proteins from the major cytochrome P450 (CYP) subfamilies were detected in all ruminant species by Western blotting, using polyclonal antibodies raised against rat or human CYP enzymes. The immunochemical data seem to suggest that humans and wild ruminants share some similar hepatic CYP enzymes corresponding to members of subfamilies 2 and 3; ruminant liver samples also contained two proteins cross-reacting with anti-rat CYP1A antibodies. High activities of CYP1A enzymes found in liver microsomes of male fallow deer and roe deer are indicative of increased susceptibility of these species towards promutagens that are metabolically activated by these CYPs. On the other hand, low activities of CYP1A-dependent alkoxyresorufin O-dealkylase activities were detected in male mouflons. Oxidative metabolism of testosterone was significantly higher in wild ruminants than the values previously reported from bulls. Androstene-3,17-dione and 6β-hydroxytestosterone were the most important products of testosterone oxidation in liver microsomes of all the ruminant species under study. The highest CYP3A-dependent testosterone 6β-hydroxylase activity was found in mouflons and fallow deer. A different pattern of CYP activities towards testosterone was found in roe deer, which showed high activities of testosterone 2β-hydroxylase and lower production of androstene-3,17-dione. An increased activity of CYP4A-dependent laurate 12-hydroxylase found in roe deer and mouflons might indicate a higher metabolic turnover of fatty acids. The data on CYP activities indicated that high metabolic rates of steroids, fatty acids, and xenobiotics may occur in male wild ruminants. The highest hepatic activities specific for CYP3A, CYP2C, CYP2D, and CYP2E enzymes were found in mouflon, suggesting that this species has the highest biotransformation capacity.
Keywords: CYPs; Ruminants; Immunoblotting; Biotransformation
Glucuronidation patterns of common urinary and serum monoester phthalate metabolites by Manori J. Silva; Dana B. Barr; John A. Reidy; Kayoko Kato; Nicole A. Malek; Carolyn C. Hodge; Donald Hurtz III; Antonia M. Calafat; Larry L. Needham; John W. Brock (pp. 561-567).
Metabolism of most diesters of phthalic acid in humans occurs by an initial phase I biotransformation in which phthalate monoesters are formed, followed by a phase II biotransformation in which phthalate monoesters react with glucuronic acid to form their respective glucuronide conjugates. The phase II conjugation increases water solubility and facilitates urinary excretion of phthalate, and reduces the potential biological activity because the putative biologically active species is the monoester metabolite. In this study, we report percentages of glucuronidation of four common phthalate monoesters, monoethyl (mEP), monobutyl (mBP), monobenzyl (mBzP), and mono-2-ethylhexyl phthalate (mEHP) in a subset of urine (mEP n=262, mBP n=283, mBzP n=328, mEHP n=119) and serum (mEP n=93, mBP n=149, mEHP n=141) samples from the general US population. The percentages of free and conjugated monoester excreted in urine differed for the various phthalates. For the more lipophilic monoesters (i.e., mBP, mBzP, and mEHP), the geometric mean of free monoester excretion ranged from 6 to 16%. The contrary was true for the most hydrophilic monoester, mEP, for which about 71% was excreted in urine as its free monoester. Furthermore, percentages of free and conjugated monoesters were similar for mEP, mBP and mEHP among serum and urine samples. Serum mBzP was largely below the method limit of detection. Interestingly, the serum mEP and mBP levels were less than 3% and 47%, respectively, of their urinary levels, whereas the level of mEHP was similar both in urine and serum.
Keywords: Serum Phthalates; Urinary phthalates; Monoethyl phthalate; Monobutyl phthalate; Monobenzyl phthalate; Mono-2-ethylhexyl phthalate; Phthalate metabolism; Phthalate glucuronidation
Gene expression in the liver of Long-Evans cinnamon rats during the development of hepatitis by Dominik Klein; Josef Lichtmannegger; Matthias Finckh; Karl H. Summer (pp. 568-575).
The Long-Evans cinnamon (LEC) rat, an authentic model for Wilson disease, is characterized by a mutation in the Atp7b gene leading to a defective copper excretion and, as a consequence, to an accumulation of the metal in the liver and copper-associated hepatotoxicity. In the present communication expression profiles of genes in the liver from wild-type Long-Evans agouti (LEA) and LEC rats at different stages of copper accumulation and liver disease were investigated. Disease states were defined according to serum aspartate aminotransferase activity and bilirubin levels in serum and from histopathology of the liver. Gene expression was determined with the Affymetrix RTU34 oligonucleotide array covering 1031 genes. Compared to the LEA rat, the nondiseased LEC rat with already increased hepatic copper level showed an enhanced expression of genes, particularly related to oxidative stress and DNA damage. During the progression of the liver disease, in particular genes related to oxidative stress, DNA damage, apoptosis and inflammation with acute-phase reaction were upregulated.
Keywords: Long-Evans cinnamon rat; Long-Evans agouti rat; Gene expression; Copper; Oligonucleotide array
Effects of daily stress or repeated paraoxon exposures on subacute pyridostigmine toxicity in rats by Jamaluddin Shaikh; Subramanya Karanth; Dibyendu Chakraborty; Steve Pruett; Carey N. Pope (pp. 576-583).
Pyridostigmine (PYR) is a carbamate cholinesterase (ChE) inhibitor used during the Persian Gulf War as a pretreatment against possible chemical nerve agent attack. Because of its quaternary structure, PYR entry into the central nervous system is limited by the blood-brain barrier (BBB). Following reports of unexplained illnesses among Gulf War veterans, however, central nervous system effects of PYR have been postulated through either stress-induced alteration of BBB permeability or via interactions with other neurotoxic agents. We evaluated the effects of daily physical (treadmill running) stress or daily exposure to a subclinical dosage of the organophosphate ChE inhibitor paraoxon (PO) on ChE inhibition in blood, diaphragm and selected brain regions in young adult male Sprague-Dawley rats following subacute PYR exposures. In physical stress studies, rats were placed on a treadmill for 90 min each day for 14 days just prior to PYR (0, 3, or 10 mg/kg per day) administration. In PO–PYR interaction studies, rats were treated with PO (0, 0.05, or 0.1 mg/kg per day) 1 h prior to daily PYR (0 or 3 mg/kg per day) administration for 14 consecutive days. Rats were evaluated daily for signs of cholinergic toxicity and were killed 1 h after the final PYR treatment. Forced running increased plasma corticosterone levels throughout the experiment (on days 1, 3, 7 and 14) when measured immediately after termination of stress. PYR-treated rats in the high dosage (10 mg/kg per day) group exhibited slight signs of toxicity (involuntary movements) for the first 6 days, after which tolerance developed. Interestingly, signs of cholinergic toxicity following PYR were slightly but significantly increased in rats forced to run on the treadmill prior to dosing. ChE activities in whole blood and diaphragm were significantly reduced 1 h after the final PYR challenge, and ChE inhibition in diaphragm was significantly greater in stressed rats than in non-stressed controls following high dose PYR (10 mg/kg per day). No significant effects of treadmill running on PYR-induced ChE inhibition in brain regions were noted, however. Repeated subclinical PO exposure had no apparent effect on functional signs of PYR toxicity. As with repeated treadmill running, whole blood and diaphragm ChE activities were significantly reduced 1 h after the final PYR administration, and ChE inhibition was significantly greater with combined PO and PYR exposures. Brain regional ChE activity was significantly inhibited after daily PO exposure, but no increased inhibition was noted following combined PO and PYR dosing. We conclude that, while some stressors may under some conditions affect functional signs of toxicity following repeated pyridostigmine exposures, these changes are likely to occur via alteration of peripheral cholinergic mechanisms and not through enhanced entry of pyridostigmine into the brain.
Keywords: Repeated stress; Pyridostigmine; Blood-brain barrier; Gulf War illnesses; Acetylcholinesterase inhibition
Embryo-lethal and teratogenic effect of the new platinum compound DPR in pregnant mice by Emanuela Ognio; Maddalena Lapide; Massimo Ottone; Václav Mandys; Miroslav Peterka; Brunella Parodi; Maurizio Viale (pp. 584-590).
Embryo-lethal and teratogenic effects caused by the cisplatin–procaine complex cis-diaminechloro-[2-(diethylamino) ethyl 4-amino-benzoate, N 4]-chlorideplatinum(II) monohydrochloride monohydrate (DPR) were examined in CD-1 mice after a single administration of 7, 14, 21 or 28 mg/kg, injected on day 6, 9, 13 or 16 of pregnancy. At day 18 of pregnancy fetuses were removed and carefully examined for external, visceral and skeletal malformations under a dissecting microscope. A significant reduction of maternal weight gain was observed in pregnant mice after the administration of 21 (day 13) or 28 mg/kg (days 9 and 13) DPR. The exposure to DPR during the organogenesis and early histogenesis periods of prenatal development (administration on day 9 or 13) induced a significant reduction of the mean percentage of live fetuses and a significant increase of the mean percentage of dead and resorbed fetuses. A dose-dependent reduction of fetal body weight was observed in surviving specimens exposed to DPR on embryonic day 9, 13 or 16. The analysis of surviving fetuses killed on day 18 of gestation showed that a few, but statistically significant, external malformations and visceral anomalies were observed after administration of 21 or 28 mg/kg DPR on embryonic day 13. External malformations consisted of three hepato-omphalocele and six palatoschisis (one random palatoschisis was also observed at 21 mg/kg DPR given on day 9), while visceral anomalies included only renal pelvis dilatation. Skeletal anomalies affected fetuses independently of the day of treatment and were more frequent at the highest doses of DPR. They consisted of a delay in skull ossifications, vertebral and sternal anomalies, and formation of extra ribs. A low and non-significant incidence of skeletal malformations (assymetric sternum) was noticed in fetuses. Our data demonstrated that DPR can cause embryotoxic effects if administered during the period of organogenesis and early histogenesis. Beside embryo-lethality, DPR induced growth retardation and malformations in surviving fetuses.
Keywords: DPR; Embryotoxicity; Teratogenicity; Mouse
Acetaldehyde adducts in the brain of alcoholics by Kazuhiko Nakamura; Kazuhiko Iwahashi; Aizo Furukawa; Kiyoshi Ameno; Hiroshi Kinoshita; Iwao Ijiri; Yoshimoto Sekine; Katsuaki Suzuki; Yasuhide Iwata; Yoshio Minabe; Norio Mori (pp. 591-593).
Acetaldehyde binds to some proteins and becomes a Schiff base. It is assumed that after the consumption of ethanol the acetaldehyde binds to the proteins to form adducts, and such acetaldehyde adducts are associated with organ diseases. We investigated the detection of acetaldehyde adducts in the brain region of a human alcoholics. Brain samples collected from an alcoholic autopsied case were used. Determination of acetaldehyde adducts was performed using a fluorescence immmunohistochemical staining method with antibodies against acetaldehyde adducts. We demonstrated acetaldehyde adducts in the frontal cortex and the midbrain of an alcoholics. Our studies showed that an acetaldehyde adduct was produced in the brain of alcoholics.
Keywords: Acetaldehyde adduct; Alcoholics; Brain
Effects of chlorohydroxyfuranones on 3-methylcholanthrene-induced neoplastic transformation in the two-stage transformation assay in C3H 10T1/2 cells by Mariitta Laaksonen; Jorma Mäki-Paakkanen; Leif Kronberg; Hannu Komulainen (pp. 594-600).
3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) are chlorination byproducts in disinfected drinking water. These compounds are positive in genotoxicity tests in vitro. We have previously shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) can induce malignant transformed foci in the two-stage cell transformation assay in C3H 10T1/2 cells in vitro in both the initiation and promotion phases. In the present study we compared the effects of CMCF, MCF and MCA in the same assay. C3H 10T1/2 mouse embryonic fibroblasts were exposed to these chlorohydroxyfuranones (CHFs) at three different concentrations in the initiation phase or the promotion phase of the assay. In the latter experiments 3-methylcholanthrene (MC, 5 µg/ml) was used as the initiating chemical. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.3 µg/ml) was used as a positive control promoter. At the end of the assay (6 weeks from the start), the transformation foci were counted and scored after fixation and staining of the cells. When added at the initiation phase of the assay on their own, CMCF and MCF, but not MCA, increased the transformation foci formation. TPA added in the promotion phase did not modify the responses of CMCF and MCF but TPA increased the number of foci in MCA-treated cells. When CHFs were added during the promotion phase to the MC-initiated cells, MCF and MCA enhanced the development of the transformation foci. The effect of CMCF was equivocal since at higher concentrations CMCF actually decreased the number of the MC-induced foci. Including the previous data for MX in this assay and considering the lowest active concentrations, the initiation activity of the foci formation decreased in the order MX >CMCF >MCF, i.e. with the decreasing number of chlorine atoms of the methyl group in the 4-position of the CHF molecule (two, one, and zero, respectively). In contrast, the activity in the promotion phase did not follow the same pattern. MX, MCF and MCA were all active over the same concentration range. Hence, in addition to MX, MCF and MCA may also possess some potential to promote tumor development.
Keywords: Tumor promotion; Chlorinated drinking water; 3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF); 3-Chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF); 3,4-Dichloro-5-hydroxy-2(5H)-furanone (MCA); Cell transformation assay
Report of accidental CS ingestion among seven patients in central Israel and review of the current literature by I. Solomon; I. Kochba; E. Eizenkraft; N. Maharshak (pp. 601-604).
A report of seven people who accidentally drank a juice contaminated with CS (o-chlorobenzylidene malononitrile) is given. Due to its mucosal irritating properties, CS (also known as "tear gas") is commonly used by policemen and soldiers in riot control. However, only a few reports of its ingestion by humans exist. Ingestion of CS may cause immediate irritation of the oral mucosa and gastrointestinal symptoms later on. Damage of internal organs, which has been shown in animals but only rarely in humans, is probably related to the dose ingested. The extensive use of CS gas merits recognition of the signs and symptoms of its exposure in order to reduce anxiety in both patients and medical staff and to facilitate fast and efficient management.
Keywords: Ingestion; CS; O-chlorobenzylidene malononitrile; Tear gas