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Archives of Toxicology (v.71, #4)

Chlorodibromomethane metabolism to bromide and carbon monoxide in rats by Dieter Pankow; Britt Damme; U. Wünscher; Kathrin Bergmann (pp. 203-210).

The chlorodibromomethane (CDBM) metabolites bromide and CO were analysed as bromide level in plasma and carboxyhaemoglobin (COHb) level in blood of rats, respectively. The mean basic levels of bromide in plasma of rats receiving vehicle were 0.075 ± 0.036 mmol/l (n = 27). After administration of CDBM at 0.4, 0.8, 1.6, and 3.1 mmol/kg p.o., the mean bromide levels rose to maximal values that were higher by a factor 27, 48, 69, and 135, respectively. Bromide elimination was slow and the plasma level was significantly increased following repeated administration in comparison to a single administration of CDBM. The CDBM concentrations in blood and in fat tissue 6 h after the last of 7 administrations of 0.8 mmol CDBM/kg p.o., once a day for 7 consecutive days, were significantly lower than 6 h after a single gavage of this CDBM dose. The mean normal level of 0.45 ± 0.32% COHb in rats (n = 30) was significantly increased following oral CDBM uptake. Initially higher COHb levels were measured after 7 consecutive applications of 0.8␣mmol/kg CDBM. After a single administration of CDBM the level of glutathione disulphide in the liver was significantly increased; this effect was reversible. The oxidative CDBM metabolism was influenced by the glutathione (GSH) concentration in the liver. The rate of COHb and bromide formation was decreased after GSH depletion due to pretreatment of rats with buthionine sulphoximine (BSO) and increased following enhancement of the GSH concentration due to pretreatment of the animals with butylated hydroxyanisole (BHA). CDBM is a substrate for cytochrome P-450 2E1 (CYP2E1), as demonstrated by the inhibition of bromide and COHb formation due to simultaneous administration of CDBM and the CYP2E1 inhibitor diethyldithiocarbamate (DDTC); also by the initially higher levels of bromide in plasma and COHb in blood after gavage of CDBM pretreated with isoniazid (INH), an inducer of CYP2E1. The increase of bromide formation after CDBM administration in phenobarbital (PB)-pretreated rats indicated that cytochrome P-450 2B1 and 2B2 (CYP2B1 and CYP2B2) play a role as catalysts of the CDBM biotransformation. It is shown that m-xylene pretreatment, which activates CYP2E1 as well as CYP2Bs, leads to a higher bromide level after CDBM administration than the INH or PB pretreatment. In liver microsomes of rats treated with CDBM (0.8 mmol/kg p.o., seven daily applications), the p-nitrophenol hydroxylase (p-NPH) activity, a marker of CYP2E1, was increased. It is concluded that CDBM may be an inducer of CYP2E1. These results combined with literature data demonstrate that the oxidation of CDBM was catalysed mainly by CYP2E1 and CYP2Bs and that there may be a risk of bromide accumulation following repeated uptake of the trihalomethane.

Keywords: Key words Chlorodibromomethane ; Bromide ; Carbon monoxide ; p-Nitrophenol hydroxylase ; Glutathione

Altered profile of urinary arsenic metabolites in adults with chronic arsenicism A pilot study by L. M. Del Razo; G. G. García-Vargas; A. Albores; H. Vargas; M. E. Gonsebatt; R. Montero; P. Ostrosky-Wegman; M. Kelsh; M. E. Cebrián (pp. 211-217).

Relationships between alterations in the profile of urinary arsenic (As) species and the presence of cutaneous signs of arsenicism were studied in Region Lagunera, Mexico. The use of urinary concentrations of putative substrates and products of the As metabolism pathway, as indicators of metabolic efficiency is also discussed. Arsenic was determined by hydride generation atomic absorption spectrophotometry and separation of As species was performed by ion exchange chromatography. The exposed group had an average of 0.408 mg As/l of total As (TAs) in their drinking water, whereas `control' individuals had 0.031 mg/l. Urinary concentrations of arsenic species and TAs were 20 to 95 times higher in the exposed group. Significant increases in the relative proportions of inorganic arsenic (As_i) and monomethylarsonic acid (MMA), accompanied by decreases of dimethylarsinic acid (DMA) were also found in exposed individuals. Therefore, significant decreases in the value of the MMA/As_i, DMA/MMA and DMA/As_i ratios were observed, suggesting a decreased As methylating ability. Exposed individuals bearing cutaneous signs had a significantly longer time of exposure, higher urinary concentrations and proportions of MMA and MMA/As_i values, and significantly lower DMA/MMA than exposed individuals without cutaneous signs. Further research is needed to identify better parameters for assessing the efficiency of As metabolism in chronically exposed populations and to confirm the potential relationship between metabolic alterations and overt signs of As toxicity.

Keywords: Key words Arsenic ; Methylation ; Chronic exposure ; Urine ; Skin diseases

Effect of probenecid on the transport of methyl mercury in erythrocytes by the organic anion transport system by Guang Wu (pp. 218-222).

The uptake of methyl mercury (MeHg) by isolated erythrocytes from rats was studied at 5° and 20 °C. The Na⁺ ion was used to examine exerted effects of probenecid on the uptake of MeHg through the organic anion transport system. Different extracellular pH levels were used to examine effects on the uptake of MeHg and on probenecid induced effects on the uptake of MeHg through the organic anion transport system; the effects of three anisotonic conditions were also determined. The results showed: (1) probenecid might partially change the role of Na⁺ ion from inhibition to stimulation for the uptake of MeHg; (2) the organic anion transport system for the uptake of MeHg was pH-dependent within the physiological range of extracellular pH; (3) the organic transport system for the uptake of MeHg was independent of deformation of the erythrocyte membrane.

Keywords: Key words Erythrocyte ; Methylmercury-glutathione ; Organic anion transport system ; Probenecid ; Uptake

Influence of the route of administration and the chemical form (MnCl2, MnO2) on the absorption and cerebral distribution of manganese in rats by H. Roels; G. Meiers; M. Delos; I. Ortega; R. Lauwerys; J. P. Buchet; D. Lison (pp. 223-230).

The absorption and cerebral distribution of manganese (Mn) have been studied with respect to the route of administration and the chemical form of the Mn compound. Different groups of adult male rats received either MnCl₂ · 4H₂O or MnO₂ once a week for 4 weeks at a dose of 24.3 mg Mn/kg body wt. (b.w.) by oral gavage (g.) or 1.22 mg Mn/kg b.w. by intraperitoneal injection (i.p.) or intratracheal instillation (i.t.). Control rats were treated with 0.9% saline. Four days after the last administration the rats were killed and the concentration of Mn measured in blood, hepatic and cerebral tissues (cortex, cerebellum, and striatum). The liver Mn concentration was not affected by the treatments whatever the chemical form or the route of administration of the Mn compound. Administration of MnCl₂ by g., i.p., and i.t. routes produced equivalent steady-state blood Mn concentrations (about 1000 ng Mn/100 ml), representing increases of 68, 59, and 68% compared with controls, respectively. Mn concentrations were significantly increased in the cortex but to a lesser extent (g., 22%; i.p., 36%; i.t., 48%) and were higher in the cerebellum after i.p. and i.t. administrations than after oral gavage. Rats treated i.t. with MnCl₂ showed an elective increase of the striatal Mn concentration (205%). In contrast, MnO₂ given orally did not significantly increase blood and cerebral tissue Mn concentrations; the low bioavailability is most likely due to the lack of intestinal resorption. Administration of MnO₂ i.p. and i.t., however, led to significant increases of Mn concentrations in blood and cerebral tissues. These increments were not significantly different from those measured after MnCl₂ administration, except for striatal Mn after i.t. which was markedly less (48%) after MnO₂ administration. A comparison of the blood Mn kinetics immediately after g. and i.t. treatment with MnCl₂ or MnO₂ indicated that the higher elevation of blood Mn concentration ( > 2000 ng Mn/100 ml) after i.t. administration of MnCl₂ could account for the elective uptake of Mn in the striatum observed in repeated dosing experiments. It is concluded that the modulation of Mn distribution in brain regions according to the route of administration and the chemical form of the Mn compound may be explained on the basis of different blood Mn kinetics and regional anatomic specificities of the striatal region.

Keywords: Key words Brain ; Manganese chloride ; Manganese dioxide ; Route of administration ; Bioavailability

Vitamin E protection of cell morphology under oxidative stress is related to cytoskeletal proteins in rat hepatocytes by Jyh-Huang Kuo; Haw-Wen Chen; Rong-Ghi R. Chou; C.-K. Lii (pp. 231-237).

A significant change in cell morphology was observed in hepatocytes treated with t-butyl hydroperoxide (t-BH). This morphological change of multiple bleb formation on cell plasma membranes was related to cell damage, and the subsequent rupture of these blebs resulted in cell death. In cells incubated with α-tocopherol before t-BH treatment, bleb formation was significantly inhibited. Using fluorescence microscopy, actin organization was shown to be related to α-tocopherol status as demonstrated by early changes in the actin network of cells in the absence of α-tocopherol. Results from SDS-polyacrylamide gel elecrophoresis further indicated that, under oxidative stress, actin molecules (45 kDa) decreased in amount and were accompanied by the formation of high molecular weight molecules. In the presence of the thiol reducing agent, dithiothreitol, both the decrease in monomeric actin and formation of high molecular weight molecules disappeared. The loss of actin showed a time-dependent response and could be observed after 15 min with t-BH treatment either in the presence or absence of α-tocopherol; however the extent was much more significant in cells with no α-tocopherol. Depletion of total membrane protein thiols was also related to vitamin E and was greater in cells with no α-tocopherol. The amount of cell damage, as determined by lactate dehydrogenase (LDH) leakage in cells with t-BH treatment over 120 min was decreased in the presence of α-tocopherol compared with the rapid increase of LDH leakage in the absence of α-tocopherol. These results indicate that vitamin E protection of cell morphology under oxidative stress is related to actin, with thiol groups in actin probably playing a key role.

Keywords: Key words Vitamin E ; Cell surface blebs ; Actin ; Hepatocytes ; Rat

Effects of n-hexane and its metabolites on cloned voltage-operated neuronal potassium channels by Michael Madeja; Norbert Binding; Ulrich Mußhoff; Ute Witting; Erwin-Josef Speckmann (pp. 238-242).

In order to study the mechanisms of acute n-hexane␣intoxication, the effects of n-hexane and its metabolites 2-hexanol, methyl-n-butyl ketone, 2,5-hexanediol and 2,5-hexanedione on the cloned voltage-operated potassium channels Kv1.1, Kv1.4, Kv2.1 and Kv3.4 were investigated with electrophysiological techniques in the expression system of Xenopus oocytes. n-Hexane had no effect at any channel, whereas some of its metabolites led to reductions of the potassium currents. The greatest effects obtained were caused by 2-hexanol at the Kv2.1 channel, resulting in reductions of 13% at 0␣mV with a concentration of 500 mg/l and IC₅₀ of ca. 3500 mg/l. The reduction appeared to be caused by a shift of the current-voltage relation to the right. Methyl-n-butyl ketone showed smaller effects, whereas 2,5-hexanedione and 2,5-hexandiol were nearly ineffective. Concerning the different potassium channels, the sensitivity to the metabolites differed. The metabolites showed greatest sensitivity towards the Kv2.1 channel and lowest sensitivity towards the Kv3.4 channel. Since the n-hexane metabolite concentrations in the brain during acute n-hexane intoxication are unknown, the relevance of the data is still unclear. The size of the effects and the currently available data on tissue concentration, however, make it more likely that the action of n-hexane and its metabolites on voltage-operated potassium channels is not a major mechanism for acute neurotoxicity.

Keywords: Key words n-Hexane ; Hexane metabolites ; Potassium current ; Oocyte

Effect of sulphur mustard on the expression of urokinase in cultured 3T3 fibroblasts by M. Detheux; H. Jijakli; D. Lison (pp. 243-249).

The expression of plasminogen activator (PA), a serine proteinase involved in the degradation of extracellular matrix proteins, has been investigated in 3T3 fibroblasts after in vitro exposure to sulphur mustard (SM). Expression of the cell-associated enzyme has been assessed with a synthetic substrate assay and at the mRNA level. Twenty-four hours after 100 μM SM, cell viability (monitored by MTT assay) was not significantly affected, but protein synthesis (tritiated leucine incorporation) was reduced to < 20% of the control value. PA activity was significantly increased compared to control cells with a 20-fold increase after 24 h. This up-regulation was independent of the cell density, occurred maximally between days 1 and 4 and persisted for at least 6 days after exposure. Lower concentrations of SM (≤ 10 μM) did not significantly affect PA activity. Northern blotting experiments revealed an increased expression of urokinase (u-PA) transcripts in cells treated with 100 μM SM, with a peak at 10 h after exposure. Conditioned culture medium from cell cultures treated with 100 μM SM did not affect the expression of PA activity in naive or SM-treated cultures. Thiodiglycol (100 μM), the main metabolite of SM, did not influence the expression of PA in the same system. Different compounds were tested for modulation of the PA up-regulation after SM exposure. Nicotinamide (5 mM), vitamin D3 (10⁻¹⁰M), extracellular calcium (2 mM) or EGTA (5 mM) had no effect. Ryanodine (10 μM) amplified the PA up-regulation by a factor of 2 and vanadate (500 μM) reduced it by ˜ 50%. Dexamethasone (1 μM) added directly after SM treatment almost completely prevented the induction of PA at both the protein and mRNA levels. Overall these results demonstrate an up-regulation of urokinase in 3T3 fibroblasts after treatment with SM, which is possibly mediated by intracellular calcium mobilization. Further studies are needed to identify the significance of this proteolytic response in the pathogenesis of blistering and/or DNA repair mechanisms.

Keywords: Key words Sulphur mustard; Urokinase; Plasminogen activator; Fibroblast; Dexamethasone

Early Effects of CI-924 on hepatic peroxisome proliferation, microsomal enzyme induction, PCNA, and apoptosis in B6C3F1 mice and Wistar rats by A. H. Hofstra; Lena M. King; Robin M. Walker (pp. 250-257).

The lipid lowering agent 5,5′{[1, 1′-biphenyl]-2,5-diylbis(oxy)}bis[2, 2-dimethylpentanoic acid] (CI-924) is a peroxisome proliferator in rats and mice, but increased the incidence of hepatic tumors in mice only. Male and female B6C3F1 mice and albino Wistar rats were treated with CI-924 at doses of 0, 25 and 75 mg/kg for 1, 3, 7 and 28 days. Our aim was to identify species differences potentially related to tumorigenicity and to establish the time course of early events related to or associated with peroxisome proliferation. After 24 h of exposure to CI-924 in the diet there were increases in carnitine acyl transferase and CYP4A1 activity in mice at 25 and 75 mg/kg. In rats, carnitine acyl transferase activity was increased after 24 h and CYP4A1 activity increased after 3 days at 75 mg/kg. Acyl CoA oxidase activity was increased at both doses in male and female rats and mice by 3 days. In general the changes in enzyme activity were of greater magnitude in rats. In contrast to the rapid peroxisome proliferation, increases in the amount of PCNA were observed in CI-924 treated rats and mice at later times after administration and only at 75 mg/kg. PCNA was increased to a similar extent in both rats and mice, while apoptosis was decreased at both doses of CI-924 after 3 days in female rats, 7 days in male rats, and was largely unchanged in mice. It was concluded that the sequence of peroxisome proliferation was generally similar in rats and mice. Early changes in cell proliferation and programmed cell death were not directly correlated with subsequent CI-924-induced hepatotumorigenicity.

Keywords: Key words Peroxisome proliferation; Species comparison; Apoptosis; Cell proliferation; CYP4A1

Postnatal development and behaviour of Wistar rats after prenatal toluene exposure by Renate Thiel; Ibrahim Chahoud (pp. 258-265).

Pregnant Wistar rats were treated with different concentrations of toluene by inhalation (300, 600, 1000 and 1200 ppm) from day 9 to day 21 of pregnancy for 6 h a day in a whole-body inhalation chamber (controls inhaled fresh air only). From day 22, rats were kept single-caged and were allowed to deliver. Besides a detailed evaluation of the physical development of the offspring we performed the following tests: forelimb-grasp reflex, righting reflex, cliff-drop aversion reflex, maintainance of balance on a rotating rod, measurement of locomotor activity and learning ability in a discrimination learning test. A toluene exposure of 1200 ppm resulted in a reduced body weight of rat dams and offspring and a higher mortality until weaning. The physical development (incisor eruption, eye opening and vaginal opening) was retarded in this group. There were no clear-cut and concentration-dependent differences in the development of reflexes, rota rod performance and locomotor activity between the offspring of animals exposed to toluene and the controls. Likewise, no effects were found on learning ability in the operant conditioning task. Compared to the controls there were no differences in mating, fertility and pregnancy indexes in the F₁-generation. The tests performed have provided no evidence that toluene exposures ≤ 1200 ppm induce adverse effects on the behaviour of rat offspring exposed during late embryonic and fetal development.

Keywords: Key words Toluene ; Postnatal development ; Behaviour

Role of cytochromes P450 in the metabolism of methyl tert -butyl ether in human livers by Jun-Yan Hong; Chung S. Yang; Maojung Lee; Yong-Yu Wang; Wei-qun Huang; Yizheng Tan; Christopher J. Patten; Flordeliza Y. Bondoc (pp. 266-269).

Methyl tert-butyl ether (MTBE) is widely used as a gasoline oxygenate for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health is a major public concern. However, information on the metabolism of MTBE in human tissues is lacking. The present study demonstrates that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and a marker for exposure to MTBE. The activity is localized in the microsomal fraction (125 ± 11 pmol TBA/min per mg protein, n = 8) but not in the cytosol. This activity level in human liver microsomes is approximately one-half of the value in rat and mouse liver microsomes. Formation of TBA in human liver microsomes is NADPH-dependent, and is significantly inhibited by carbon monoxide (CO), an inhibitor of cytochrome P450 (CYP) enzymes, suggesting that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Both CYP2A6 and 2E1 are known to be constitutively expressed in human livers. To examine their involvement in MTBE metabolism, human CYP2A6 and 2E1 cDNAs were individually co-expressed with human cytochrome P450 reductase by a baculovirus expression system and the expressed enzymes were used for MTBE metabolism. The turnover number for CYP2A6 and 2E1 was 6.1 and 0.7 nmol TBA/min per nmol P450, respectively. The heterologously expressed human CYP2A6 was also more active than 2E1 in the metabolism of two other gasoline ethers, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME). Although the contributions of other human CYP forms to MTBE metabolism remain to be determined, these results strongly suggest that CYP enzymes play an important role in the metabolism of MTBE in human livers.

Keywords: Key words Methyl tert-butyl ether ; Metabolism ; Human liver microsomes ; Cytochromes P450

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Archives of Toxicology (v.71, #4)