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Archives of Toxicology (v.81, #3)
Effect of co-exposure to lead and cadmium on antioxidant status in rat ovarian granulose cells by Laxmipriya P. Nampoothiri; Avnika Agarwal; Sarita Gupta (pp. 145-150).
The effects of combined exposure to lead and cadmium on granulose cells were studied. Adult female rats were treated i.p. with either lead acetate (LA) or cadmium acetate (CA) both, alone, or in combination at a dose of 0.05 mg/kg body weight on a daily basis for 15 days. Both metals were accumulated in the ovary after metal exposure. Metal exposure caused a decrease in reduced glutathione content along with elevated lipid peroxidation in all groups. Granulose cells of both cadmium as well as combination group demonstrated a maximum increase in lipid peroxides and catalase activity, along with decreased glutathione status and superoxide dismutase activities. Combined treated animals exhibited an intermediate effect in antioxidant status. However, “in vitro” exposure showed no significant change in antioxidant enzymes in all metal exposed cells. Data from the present study indicates that lead and cadmium in isolation and in combination cause oxidative stress. Lead and cadmium in combination do not show additive or synergistic effect indicating the competition between them due to similarity in electronic affinities. Present study highlights the effects of toxic metals that disturb membrane integrity of cells via ROS and thereby classifying mechanism for altered receptor binding, steroidogenesis, and hormone production.
Keywords: Lead; Cadmium; Antioxidant status; Granulose cells
Tissue distribution and excretion of myosmine after i.v. administration to Long–Evans rats using quantitative whole-body autoradiography by Susanna Glas; Stefan Tyroller; Wolfgang Zwickenpflug; Kurt Steiner; Gudrun Kiefer; Elmar Richter (pp. 151-161).
Occurrence of the tobacco alkaloid myosmine has been proven in various staple foods, vegetables and fruits. Myosmine can be easily activated by nitrosation yielding 4-hydroxy-1-(3-pyridyl)-butanone (HPB) and the esophageal carcinogen N′-nitrosonornicotine. Most of the reaction products after myosmine peroxidation were also identified as urinary metabolites after oral administration to rats. Whole-body autoradiography with freeze dried or multiple solvent extracted tissue sections was used to trace [2′-14C]myosmine (0.1 mCi/kg bw) 0.1, 0.25, 1, 4 and 24 h after i.v. injection in Long–Evans rats. In addition, in vitro binding of radioactivity to esophageal and eye tissue was determined and excretion of radioactivity via urine and feces was quantified. Radioactivity is rapidly eliminated by renal excretion. Approximately 30% of the administered radioactivity was recovered in urine within the first 4 h and excretion with urine (72%) and feces (15%) was nearly complete after 24 h. A rapid concentration of radioactivity can be seen in the stomach and in the salivary and lachrymal glands. Rats killed 1 and 4 h after treatment showed by far the highest labeling in the accessory genital gland. High levels of nonextractable radioactivity were present in esophageal tissue and melanin. The half lives for the disappearance of radioactivity from various tissues are in the order of about 1 h. Eye and esophagus sections both showed nonextractable labeling after in vitro incubation with 14C-myosmine. In conclusion, the toxicological significance of myosmine accumulation in esophagus and accessory genital gland requires further investigations. Hair analysis might be applicable for myosmine biomonitoring, because of possible enrichment in melanin containing tissues.
Keywords: Myosmine; Tobacco alkaloid; Tissue distribution; Excretion; Long–Evans rat; Quantitative whole-body autoradiography
Identification of the human liver UDP-glucuronosyltransferase involved in the metabolism of p-ethoxyphenylurea (dulcin) by Yoshihiro Uesawa; Adam G. Staines; David Lockley; Kiminori Mohri; Brian Burchell (pp. 163-168).
Dulcin (DL), now banned, was once a widely used artificial sweetener. DL possesses an ureido group that is metabolized by direct glucuronidation in rabbit liver microsomes. Dulcin N-glucuronide (DNG) is the only type of ureido N-glucuronide known to date; ureido glucuronidation in humans has not been previously reported. Accordingly, the glucuronidation of DL was studied using human liver microsomes (HLM) and expressed human UDP-glucuronosyltransferase (UGT) enzymes. The average K m and V max values from nine HLM samples were 2.10 mM and 0.156 nmol/mg/min, respectively. Of the six human UGT isoforms screened for their ability to glucuronidate DL, only UGT1A1 and UGT1A9 showed activity. The apparent K m values using UGT1A1 and UGT1A9 were 5.06 and 6.99 mM, and the apparent V max values were 0.0461 and 0.106 nmol/min/mg, respectively. Phenolphthalein, a substrate for UGT1A9, inhibited DL glucuronidation in HLM competitively (K i = 0.356 mM), but bilirubin, a substrate for UGT1A1, did not. These results suggest that UGT1A9 is a key enzyme catalyzing the glucuronidation of DL.
Keywords: Dulcin; UGT1A1; UGT1A9; Ureido group; Glucuronidation
Polycyclic aromatic hydrocarbon (PAH) metabolizing enzyme activities in human lung, and their inducibility by exposure to naphthalene, phenanthrene, pyrene, chrysene, and benzo(a)pyrene as shown in the rat lung and liver by Eivor Elovaara; Jouni Mikkola; Helene Stockmann-Juvala; Leena Luukkanen; Helena Keski-Hynnilä; Risto Kostiainen; Markku Pasanen; Olavi Pelkonen; Harri Vainio (pp. 169-182).
In order to survey changes and activities in the polycyclic aromatic hydrocarbon (PAH)-metabolizing enzymes implicated in lung cancer susceptibility studies, we investigated enzyme induction by 2–5-ring-sized ‘biomarker’ PAHs in rat liver and lung, and the activities in five human lung specimens. Naphthalene, phenanthrene, pyrene, chrysene, and benzo[a]pyrene (BaP) were administered to rats for 3 days (25–128 mg/kg/day) and the responses compared with those of model inducers. PAH treatment increased the CYP1A-catalyzed activity of pyrene 1-hydroxylation and 7-ethoxyresorufin O-deethylation in rat liver by up to 28- and 279-fold, and in rat lung by up to 22- and 51-fold, respectively. 1-Naphthol (hUGT1A6), 1-hydroxypyrene (hUGT1A6/1A9), and entacapone (hUGT1A9) are markers of PAH-glucuronidating human uridine diphosphate-glucuronosyltransferases (UGT). These activities increased up to 6.4-fold in rat liver and up to 1.9-fold in rat lung. NADPH:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase activities increased up to 5.3- and 1.6-fold (liver), and up to 4.4- and 1.4-fold (lung), respectively. CYP1A showed the best liver-to-lung relationship (R 2 = 0.90). The inducing efficiency by PAHs differed extensively: control ≤ naphthalene < phenanthrene, pyrene << chrysene < BaP. In human lung (non-smokers), the marker activities of CYP1A1, UGT1A6/1A9, and NQO1 were lower than those in rat lung. Epoxide hydrolase activity was 1,000-fold higher than the pulmonary CYP1A1 activities. Human UGT and NQO1 displayed large variations (>60-fold), many times greater than the experimental (inducible/constitutive) variation in the rat. Kinetics of 1-hydroxypyrene glucuronidation showed two low-K m forms both in rat and human lung. Since the 2–4-ring PAHs (major constituents) were poor enzyme inducers, it appears that the PAH-metabolizing pathways are mainly induced by BaP-type minor constituents. Gene–environmental interactions which magnify polymorphic variability in pulmonary bioactivation/detoxification capacity probably play a key role in individual susceptibility to (or protection against) chemically induced lung cancer. Hence, human exposure to PAH mixtures with high content of BaP-type hydrocarbons confers a potentially higher health risk than PAH mixtures with low content of procarcinogens.
Keywords: PAH metabolism; Pyrene hydroxylation; 1-Hydroxypyrene glucuronidation; Enzyme induction; UGT kinetics; Human lung; Rat lung and liver; Individual susceptibility
A comparison of the concentration–effect relationships of PAHs on CYP1A induction in HepG2 and Mcf7 cells by K. Skupinska; I. Misiewicz; T. Kasprzycka-Guttman (pp. 183-200).
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants. Some compounds belonging to this group are considered carcinogenic to people. In order to yield carcinogenic properties, these compounds must be metabolically transformed by enzymes of cytochrome P450 family to oxy-derivatives. In this study, the ability of the following six PAHs: anthracene (Ant), benz(a)anthracene (BA), naphthacene (Nap), benzo(a)pyrene (BaP), dibenz(a,c)anthracene (DB(a,c)A) and dibenz(a,h)anthracene (DB(a,h)A) to induce enzymes of cytochrome P450 (CYP450), in particular CYP1A1 and CYP1A2 in Mcf7 and HepG2 cells was studied. The induction of CYP1A enzymes was assessed at the level of enzymatic protein and enzymatic activity. The change in CYP1A1 and CYP1A2 protein level was assessed by means of confocal microscopy. The ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-deethylase (MROD) assays were applied to determine the CYP1A1 and CYP1A2 activity. The Induction Equivalency Factors (IEFs) were also determined. According to EROD and MROD assay and calculated IEFs the following order of the inducing potency was determined in HepG2 cells: DB(a,h)A > BaP > DB(a,c)A ≈ BA > Nap > Ant, and in Mcf7 cells: DB(a,h)A > DB(a,c)A > BaP > Nap > BA > Ant. The assessment of the protein levels revealed that DB(a,h)A was also the strongest inducer of protein level, however the correlation between enzymatic activity and protein level induction by other PAHs was not always evident. The EROD and MROD activities were higher in Mcf7 than in HepG2 cells, however the CYP1A2 protein level was shown to be higher in HepG2 cells. The results obtained indicate possible catalytic enzymatic activity alterations induced by PAHs.
Keywords: Anthracene; Benz(a)anthracene; Naphthacene; Benzo(a)pyrene; Dibenz(a,c)anthracene; Dibenz(a,h)anthracene; Cytochrome P450; Induction; Confocal microscopy; Ethoxyresorufin O-deethylase (EROD); Methoxyresorufin O-demethylase (MROD)
Hepatotoxin-induced hypertyrosinemia and its toxicological significance 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. 201-210).
A 1H Nuclear Magnetic Resonance (NMR) spectroscopic investigation of the effects of single doses of four model hepatotoxins on male Sprague–Dawley rats showed that hypertyrosinemia was induced by three of the treatments (ethionine 300 mg/kg, galactosamine hydrochloride 800 mg/kg and isoniazid 400 mg/kg) but not by the fourth (thioacetamide 200 mg/kg). Concomitant histopathological and clinical chemistry analyses showed that hypertyrosinemia could occur with or without substantial hepatic damage and that substantial hepatic damage could occur without hypertyrosinemia. However, in the rats dosed with galactosamine hydrochloride, which showed highly variable amounts of liver damage at ca. 24 h after dosing, a clear relationship was found between the degree of hypertyrosinemia and the extent of the hepatic necrosis induced. In line with the cause of clinically observed Type II Tyrosinemia, we consider that the critical event in the onset of hepatotoxin-induced hypertyrosinemia is likely to be a reduction in hepatic tyrosine aminotransferase (TAT) activity. We discuss mechanisms by which TAT activity could be lost with special consideration given to pyridoxal 5′-phosphate (P5P) depletion and to the inhibition of protein synthesis. This analysis may have implications for the interpretation of clinical measures of liver status such as Fischer’s ratio and the branched-chain tyrosine ratio (BTR).
Keywords: Ethionine; Galactosamine; Isoniazid; Thioacetamide; Liver; Hypertyrosinemia; Tyrosine Aminotransferase; Rat
Isolation of chebulic acid from Terminalia chebula Retz. and its antioxidant effect in isolated rat hepatocytes by Hyun-Sun Lee; Sung-Hoon Jung; Bong-Sik Yun; Kwang-Won Lee (pp. 211-218).
A hepatoprotective compound was isolated from the ethanolic extract of the fruits of Terminalia chebula Retz. by consecutive solvent partitioning, followed by silica gel and Sephadex LH-20 column chromatographies. The purified compound was identified as a mixture of chebulic acid and its minor isomer, neochebulic acid, with a ratio of 2:1 by spectroscopic analysis including 1D and 2D NMR and MS spectroscopy. To our knowledge, this is the first report on the protection of rat hepatocytes against oxidative toxicity by chebulic acid obtained from T. chebula Retz. This compound exhibited in vitro a free radical-scavenging activity and ferric-reducing antioxidant activity. Also, the specific ESR spectrum for the •OOH radical signals consisting of three-line ESR spectra was within the field of 0.27 mT, whereas 2.5 and 0.25 mg/ml of chebulic acid significantly reduced the signal intensity of the ESR spectra to 0.06 mT and 0.11 mT, respectively. Using isolated rat hepatocyte experiment, we demonstrated that the treatment of hepatocytes with chebulic acid significantly reduced the tert-butyl hydroperoxide (t-BHP)-induced cell cytotoxicity, intracellular reactive oxygen species level, and the ratio of GSSH, oxidized form of glutathione (GSH) to the over total GSH (GSH + GSSG) (4.42%) as compared to that with t-BHP alone (8.33%).
Keywords: Chebulic acid; Cytotoxicity; Antioxidant; Hepatocytes; Oxidative stress; Rat
Induction of peroxisome proliferator-activated receptor alpha (PPARα)-related enzymes by di(2-ethylhexyl) phthalate (DEHP) treatment in mice and rats, but not marmosets by Yuki Ito; Osamu Yamanoshita; Yoshimasa Kurata; Michihiro Kamijima; Toshifumi Aoyama; Tamie Nakajima (pp. 219-226).
To clarify species differences in the induction of peroxisome proliferator-activated receptor alpha (PPARα)-related enzymes by di(2-ethylhexyl)phthalate (DEHP) exposure, we investigated the inductions of PPARα and its target genes (mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD) and peroxisomal keto-acyl-CoA thiolase (PT) in liver from mice (CD-1), rats (Sprague–Dawley), and marmosets (Callithrix jacchus) exposed to DEHP. Male mice and rats were treated with 0, 1.25 and 2.5 mmol/kg DEHP for 2 weeks, and marmosets with 0, 0.25, 1.25 and 6.25 mmol/kg DEHP for 15 months by gavage. Hepatic mono(2-ethylhexyl)phthalate (MEHP) levels were significantly higher in mice and rats than in marmosets. The constitutive expression of hepatic PPARα was 5–7 times greater in rats and mice than in marmosets, but DEHP treatment did not induce PPARα-mRNA in all animals. The treatment-induced PT expression detected either by anti-PT antibody or PT-mRNA levels in the liver only from mice and rats, and the induction of the mRNA was greater in the latter than in the former. Thus, DEHP used in this experiment influenced the peroxisomal enzymes in mice and rats, but did not affect the mitochondrial enzymes in any animals or the peroxisomal enzymes in marmosets. These results suggest that there are species differences in the induction of PPARα-related enzymes, especially in peroxisomal enzymes by DEHP treatment, and their underlying mechanism may in part reside in the different constitutive levels of PPARα and different forming levels of MEHP.
Keywords: Species differences; Mice; Rats; Marmosets; PPARα-related enzymes