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Archives of Toxicology (v.71, #11)

UDP-GT involvement in the enhancement of cell proliferation in thyroid follicular cell proliferative lesions in rats treated with thiourea and vitamin A by Kiyoshi Takegawa; K. Mitsumori; Hiroshi Onodera; Mamoru Mutai; Keisuke Kitaura; Masakazu Takahashi; Chikako Uneyama; Kazuo Yasuhara; Tokuma Yanai; Toshiaki Masegi; Yuzo Hayashi (pp. 661-667).

The mechanisms underlying enhanced cell proliferation in thyroid proliferative lesions of rats simultaneously treated with large amounts of vitamin A (VA) and thiourea (TU) were investigated. Male F344 animals were initiated with N-bis(2-hydroxypropyl)nitrosamine (2800 mg/kg body weight, single s.c. injection). Starting 1 week later, groups received water containing 0.2% TU (TU group), diet containing 0.1% VA (VA group), both 0.2% TU and 0.1% VA (TU + VA group) or tap water/basal diet without supplement (control group) for 10 weeks. The serum levels of triiodothyronine (T3) and thyroxine (T4) were decreased and the thyroid stimulating hormone (TSH) levels were elevated in the TU and TU + VA groups, with the degree of change being significantly greater in the combined treatment group. The induction of P450 isoenzymes by TU was not enhanced by VA supplementation, but uridine diphosphate glucuronosyltransferase (UDP-GT) activity in the liver was significantly increased in the TU + VA group compared to the TU group. Thyroid weights were increased in both the TU and TU + VA groups, this being more pronounced with VA supplementation. Thyroid follicular cell hyperplasias and neoplasias were induced to similar extents in both TU treated groups, but their cell proliferation appeared to be increased by the VA supplementation. The results of the present study suggest that enhanced cell proliferation is due to increased TSH stimulation, resulting from the decrease in serum T3/T4 levels brought about by induction of liver UDP-GT activity with the combined action of TU + VA as well as inhibition by TU of thyroid hormone synthesis in the thyroid.

Keywords: Key words Thyroid carcinogenesis; Vitamin A; Thiourea; UDP-GT; Rat

l-2-Chloropropionic acid metabolism and disposition in male rats: relevance to cerebellar injury by Ian Wyatt; Michael Farnworth; Andrew J. Gyte; Edward A. Lock (pp. 668-676).

l-2-Chloropropionic acid (L-CPA) produces selective necrosis to the granule cell layer of the rat cerebellum. As part of a study to understand the mechanism of selective toxicity we have investigated the metabolism and disposition of [2-¹⁴C]L-CPA in the rat, with particular emphasis on the brain. Following a single oral non-toxic dose of 250 mg/kg or a neurotoxic dose of 750 mg/kg or 250 mg/kg per day for 3 days, L-CPA is very rapidly absorbed from the gastrointestinal tract into the blood stream. Peak plasma concentrations of 2 mM (250 mg/kg) and 6 mM (750 mg/kg) L-CPA occurred within 1 h of dosing, and the compound was readily cleared from the plasma with a half-life of 2.6 h. The only metabolite detected in the plasma was 2-S-cysteinylpropanoic acid, presumably derived from the glutathione conjugate. About 60% of the dose is excreted in the urine in the first 24 h as unchanged L-CPA, with a smaller amount excreted as the mercapturate, 2-S-N-acetylcysteinylpropanoic acid. Little radiolabel from L-CPA is excreted in the faeces; however, ∼18% of a 250 mg/kg dose of L-CPA is eliminated as carbon dioxide. The radiolabel from [2-¹⁴C]L-CPA present in the cerebellum, forebrain and liver at all time intervals examined was L-CPA. There was some indication of retention of L-CPA in the brain relative to the plasma with a small but consistently higher concentration found in the cerebellum. Whole body autoradiography studies indicated some selective retention of radiolabel in the cerebellum after the third dose of 250 mg/kg [2-¹⁴C]L-CPA. Our findings indicate that the initial insult to the cerebellum following L-CPA administration is probably due to the parent compound however, the prolonged presence of 2-S-cysteinylpropanoic acid in the plasma and concomitant depletion of glutathione in the cerebellum may also play a role in the toxicity. The relevance of the slightly greater retention of L-CPA in the cerebellum to the selective neurotoxicity of L-CPA requires further study.

Keywords: Key wordsl-2-Chloropropionic acid; Glutathione conjugation; Cerebellar granule cell necrosis

Protective effects of vitamin E and C on cisplatin nephrotoxicity in developing rats by D. Appenroth; S. Fröb; L. Kersten; F. -K. Splinter; K. Winnefeld (pp. 677-683).

The kinetics of vitamin E was followed in serum, liver and kidney of 10- and 55-day-old rats after the administration of a single i.m. dose of 100 mg α-tocopherol acetate/100 g body wt. The basal levels without vitamin E administration were significantly higher in serum and liver of 10- than 55-day-old rats. The effect of vitamin E on cisplatin (CP; 0.6 mg/100 g body wt., i.p.) nephrotoxicity was investigated by determining urinary volume and protein excretion, as well as the concentration of blood urea nitrogen (BUN) and lipid peroxides in renal tissue (LPO). Previously described age differences in CP nephrotoxicity were confirmed. The administration of vitamin E, 12 h prior to CP, diminished the toxic effect of CP in young and adult rats. This effect could not be enhanced by a second administration of vitamin E. The simultaneous administration of vitamin E and C 12 h prior to CP intensified the protective effect of a single administration of vitamin E in 10- and 55-day-old rats without influencing the concentration of platinum in renal tissue.

Keywords: Key words Cisplatin; Nephrotoxicity; Vitamin E; Vitamin C; Rats

Undernutrition during hyperoxic exposure induces CYP2E1 in rat liver by Oleg G. Khatsenko; Ram K. Sindhu; Yutaka Kikkawa (pp. 684-689).

Induction of cytochrome P450 2E1 (CYP2E1) has been shown to occur through two distinct mechanisms. The first is seen by treatment of rats with acetone, pyrazole, and 4-methyl-pyrazole, which induces CYP2E1 protein without affecting the mRNA level. The second is observed in starvation, diabetes, and obesity, in which an increase of CYP2E1 protein is associated with an increase of the CYP2E1 mRNA. It has been reported by (Tindberg and Ingelman-Sundberg 1989) that hyperoxic exposure (95% O₂) induced a several-fold increase of CYP2E1 protein in both the liver and lung of exposed rats without affecting the level of CYP2E1 mRNA. During the course of our previous study which demonstrated hyperoxia-induced specific pretranslational induction of CYP1A1/2 in the liver and CYP1A1 in the lung, we observed a progressive increase of hepatic CYP2E1 mRNA in animals of the hyperoxia group. Hyperoxia is accompanied by some degree of starvation and our earlier experiments were conducted with rats of significantly greater body weight than those used by Tindberg and Ingelman-Sundberg (260 vs 150 g). Thus we reevaluated the changes of CYP2E1 in the current study with the use of food-restricted control, and by utilizing rats of comparable weight (∼150 g) to that utilized by Tindberg and Ingelman-Sundberg. The results obtained in the present study showed that there was a significant increase in the levels of hepatic CYP2E1 mRNA, protein, and p-nitrophenol hydroxylase activity in the food-restricted control group compared to the untreated controls. Rats from the hyperoxia group also demonstrated a similar increase of these three parameters in their livers but showed no significant difference compared with the results of the food-restricted control group. Rats weighing ∼260 g were also examined with similar food restriction and hyperoxia, and the results were essentially similar to those obtained with the younger rats. The lungs of rats from food-restricted control and hyperoxia groups showed no increase of any of the CYP2E1 parameters. The results obtained in the current study, therefore, indicate that hyperoxia has no effect on CYP2E1 expression in both the liver and lung. Increased CYP2E1 mRNA, protein, and p-nitrophenol hydroxylase activity seen in the liver of rats, but not in the lungs, are consistent with the notion that undernutrition during hyperoxia is the underlying mechanism for this induction.

Keywords: Key words CYP2E1; Hyperoxia; Starvation

Absence of a differential induction of cytochrome P450 2E1 by different alcoholic beverages in rats: implications for the aetiology of human oesophageal cancer by Mathilde Lechevrel; Christopher P. Wild (pp. 690-695).

Alcohol is an important risk factor for human oesophageal cancer. There is evidence from epidemiological studies that some specific alcoholic drinks, e.g. Calvados apple brandy, are associated with a greater risk than others. Alcohol induces cytochrome P450 2E1 (CYP2E1) and the hypothesis was tested that different alcoholic beverages, containing a variety of alcoholic compounds, could differentially induce expression of cytochrome P450 enzymes. Twelve groups of five rats each were treated for 3 days with different alcoholic beverages (ethanol alone, whisky, farm-produced or commercial Calvados brandy, beer, cider, wine) adjusted to 4, 10 or 20% of ethanol in drinking water. Immunoblotting using a monoclonal antibody specific for rat CYP2E1 revealed a single protein band in liver microsomes. Densitometric quantitation of microsomal proteins demonstrated a significant two-, three- and sixfold increase in band intensity after treatment with ethanol concentrations of 4, 10 and 20% respectively, compared to control rats drinking water alone. There was a dose-dependent increase in liver microsomal metabolism of CYP2E1 substrates (para-nitrophenol and dimethylnitrosamine) in ethanol-treated rats. However, there were no significant differences in the level of CYP2E1 protein or enzymatic activity between the different alcoholic beverages at the same ethanol concentration. There was a slight increase in hepatic CYP1A-related enzymatic activities in the alcohol-treated rats compared to the controls, but no difference between the treated groups either with dose of ethanol or type of beverage. These data show that induction of CYP2E1 with acute alcohol treatment is predominantly determined by the ethanol content of the beverage.

Keywords: Key words Alcohol; Cytochrome P450 2E1; Oesophageal cancer; N-Nitrosamines

Age dependent differential effects of cigarette smoke on hepatic and pulmonary xenobiotic metabolizing enzymes in rats by Benay C. Eke; Nevin Vural; Mümtaz İşcan (pp. 696-702).

The effects of cigarette smoke (CS) on hepatic and pulmonary monooxygenase (MO) activities (aniline␣4-hydroxylase, AH; aminopyrine N-demethylase, AMND; 7-ethoxyresorufin O-deethylase, EROD; p-nitroanisole O-demethylase, p-NAOD), lipid peroxidation (LP), and reduced glutathione (GSH) levels and glutathione S-transferase (GST) activities toward several substrates (1-chloro-2,4-dinitrobenzene, CDNB; 1,2-dichloro-4-nitrobenzene, DCNB; ethacrynic acid, EAA; 1,2-epoxy-3-( p-nitrophenoxy)-propane, ENPP) were determined in 20-, 90- and 360-day-old male rats. The animals were exposed to CS five times a day, with 1 h intervals, for 3 days in a chamber supplied alternatively with smoke and fresh air, and were killed 16 h after the last treatments. The hepatic AH activity increased significantly in 20-day-old rats and remained unaltered in older age groups. The hepatic AMND activity unaltered, significantly increased and decreased in 20-, 90- and 360-day-old rats, respectively. The pulmonary AH activity increased significantly in 20- and 90-day-old rats whereas no alteration was noted in 360-day-old rats. CS was ineffective on pulmonary AMND activity at all ages. CS increased hepatic and pulmonary EROD and p-NAOD activities significantly in all age groups compared to controls. In liver, LP level was significantly increased, decreased, and unaltered in 20-, 90- and 360-day-old rats, respectively. CS increased hepatic GSH level significantly in 90-day-old rats but was not effective in the other age groups. In lung, LP level was increased in 90- and 360-day-old rats and unaltered in 20-day-old rats. CS increased pulmonary GSH level significantly in 90-day-old rats and did not have any effect in the other age groups. The hepatic GST activities toward CDNB and DCNB decreased significantly in 360-day-old rats and were unaltered in the younger age groups. The hepatic GST activity toward EAA was unaltered, significantly increased and decreased in 20-, 90- and 360-day-old rats, respectively. The hepatic GST activity toward ENPP decreased significantly in 20- and 90-day-old rats but was unaltered in the oldest group of rats. In 20-day-old rats, the pulmonary GST activity toward ENPP increased significantly whereas the other GST activities did not alter. In 90-day-old rats, however, CS significantly decreased all the pulmonary GST activities studied. Unaltered DCNB GST, significant increase in EAA GST and decrease in CDNB and ENPP GST activities of lung were noted in 360-day-old rats. These results reveal that the regulation in rats of hepatic and pulmonary MO and GST activities are differentially influenced by CS as a function of age.

Keywords: Key words Cigarette smoke; Monooxygenases; Lipid peroxidation; Glutathione; Glutathione S-transferases

Distribution studies in CD-1 mice administered [14C]muconaldehyde by Zhihua Zhang; Keith Cooper; Bernard D. Goldstein; Gisela Witz (pp. 703-708).

Trans,trans-muconaldehyde (muconaldehyde, MUC), a microsomal hematotoxic ring-opened metabolite of benzene, has been proposed to play a role in benzene hematotoxicity. In the present study, [¹⁴C]-muconaldehyde was administered to CD-1 mice and the distribution of [¹⁴C]muconaldehyde equivalents was investigated. The study was carried out to evaluate whether [¹⁴C]muconaldehyde equivalents could reach the bone marrow. [¹⁴C]Muconaldehyde at a dose of 2 mg/kg was administered intraperitoneally (3.4 μCi/mouse) and by intravenous injection (2.7 μCi/mouse). The amount of [¹⁴C]muconaldehyde equivalents was measured in the bone marrow, blood, liver, lung, kidney and spleen at 0.25, 0.5, 1, 2, 4, and 24 h after [¹⁴C]MUC administration. The results indicate that 0.044% or 0.018% of the total dose administered when given i.v. or i.p., respectively, reached the bone marrow. The elimination of the radioactivity in all organs had at least two phases. The bone marrow, kidney, and lung had a rapid first phase (t _1/2 0.5–1.2 h) and a slower second phase (t _1/2 2.8–15.7 h). In the liver, a slow first phase (t _1/2 3.7 h) was followed by a more rapid second phase (t _1/2 1.5 h). The level of radioactivity in blood and bone marrow was significantly higher when [¹⁴C]muconaldehyde was administered intravenously compared with intraperitoneally, demonstrating that the route of administration affects the distribution of [¹⁴C]muconaldehyde equivalents.

Keywords: Key words Distribution; Muconaldehyde; Benzene metabolite; Mice

Effects of ochratoxin A on DNA repair in cultures of rat hepatocytes and porcine urinary bladder epithelial cells by Angelika Dörrenhaus; Wolfram Föllmann (pp. 709-713).

In cultured rat hepatocytes the mycotoxin ochratoxin A (OTA) induced unscheduled DNA synthesis (UDS) only in a narrow concentration range. Using a culture medium supplemented with 1% fetal calf serum, at 750 nM OTA a weak induction and at 1 μM OTA a marked induction of DNA repair was observed (15 ± 11 and 38 ± 24% cells in repair, respectively). Concentrations >1 μM OTA were cytotoxic, and <750 nM no induction occurred. In cultures of cells from the urinary bladder (porcine urinary bladder epithelial cells; PUBEC), a target organ of the mycotoxin, OTA induced UDS in a concentration-dependent manner. To inhibit the proliferation of the cultured epithelial cells, which would counteract the detection of DNA repair, epidermal growth factor was omitted and an arginine-deficient medium (ADM) was used. Under these serum-free culture conditions the amount of cells undergoing DNA repair in PUBEC control cultures was ∼7±4%, a value also comparable to those of control cultures of rat hepatocytes. At concentrations between 250 nM and 1 μM OTA a concentration-dependent increase of cells in repair was observed. Above 1 μM OTA was cytotoxic. At this concentration a maximum of ∼61±9% of the cells undergo DNA repair. This amount is comparable to control cultures incubated with 5 or 10 mM ethylmethane-sulphonate (EMS) (49±9 and 69±10% cells in repair, respectively), used as a positive control. These results show that in cultured rat hepatocytes induction of UDS is relatively weak whereas in urothelial cells this effect was significant. Whether this effect is due to OTA metabolites formed locally in the urothelium cannot be excluded since PUBEC have been shown to be able to metabolize xenobiotics independently from the liver.

Keywords: Key words Ochratoxin A; Urinary bladder; DNA repair

Direct reaction of oximes with sarin, soman, or tabun in vitro by G. Becker; A. Kawan; L. Szinicz (pp. 714-718).

The direct reaction of seven pyridinium oximes with the nerve agents sarin, soman, and tabun was followed by a spectrophotometric method. The half-lives (t _1/2) of the oximes, the first- and second-order rate constants (k ₁, k ₂), and the maximal reaction velocity ( $ oundv $ _max) were calculated according to changes in the absorbance of the zwitterion (betaine) peak. In all cases the reaction velocity of the nerve agents with any of the oximes was highest with tabun, followed by sarin and then soman. Comparing the reaction rates of three therapeutically used oximes with the same nerve agent, the highest rate was obtained for soman with obidoxime, for sarin with 2-PAM, and for tabun with HI 6. The maximal reaction velocities reveal that the detoxification of the nerve agents by direct reaction with oximes and the subsequent decomposition of the phosphonyl oxime in vivo do not substantially contribute to the therapeutic effect of these antidotes.

Keywords: Key words Oximes; Sarin; Soman; Tabun; Direct reaction

A note on the physiological background of the ethylene oxide adduct 7-(2-hydroxyethyl)guanine in DNA from human blood by Hermann M. Bolt; Monika Leutbecher; Klaus Golka (pp. 719-721).

A newly developed high-performance liquid chromatography (HPLC) method involves derivatization with phenylglyoxal and fluorescence detection, using 7-methylguanine as internal standard. The physiological background of the adduct 7-(2-hydroxyethyl)guanine in DNA isolated from human blood was determined by this method. In five persons the range of the adduct was between 2.1 and 5.8 pmol/mg DNA (mean 3.2). This finding is consistent with previous data obtained by others, using different analytical methods, and points to an intrinsic carcinogenic risk due to endogenous ethylene oxide.

Keywords: Key words Ethylene; Ethylene oxide; 7-(2-Hydroxyethyl)guanine; DNA adducts

Effects of single exposure to toluene vapor on the expression of immediate early genes and GFAP gene in the mouse brain by Masato Matsuoka; Hirohiko Matsumura; Hideki Igisu; Hajime Hori; Isamu Tanaka (pp. 722-723).

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Archives of Toxicology (v.71, #11)