Protective effect of vitamin E on chromium (VI)-induced cytotoxicity and lipid peroxidation in primary cultures of rat Hepatocytes

Pretreatment of primary cultures of rat hepatocytes with α-tocopherol succinate (vitamin E) for 20 h prior to exposure to K₂Cr₂O₇ resulted in a marked decrease of chromium (VI)-induced cytotoxicity, as evaluated by the leakage of lactate dehydrogenase, without affecting cellular uptake and subcellular distribution of chromium. The levels of chromium (VI)-induced lipid peroxidation, as monitored by malondialdehyde formation, were also inhibited by pretreatment with the vitamin. Pretreatment with vitamin E normalized the levels of nonenzymatic antioxidants such as glutathione and vitamin C suppressed by dichromate, and caused a distinct accumulation of vitamin E in hepatocytes. However, vitamin E pretreatment did not affect the activities of enzymatic antioxidants including glutathione reductase, superoxide dismutase, and catalase suppressed by dichromate. These results indicate that the protective effect of vitamin E against chromium (VI)-induced cytotoxicity as well as lipid peroxidation, may be associated more with the level of nonenzymatic antioxidants than the activity of enzymatic antioxidants. Received: 31 October 1995/Accepted: 16 July 1996     

Protective role of vitamin E on nickel and/or chromium induced oxidative stress in the mouse ovary

In the present study, we report the invivo effects of nickel chloride (NiCl₂; 8 and 16 mg/kg body weight) and/or potassium dichromate (K₂Cr₂O₇; 5 and 10 mg/kg body weight) in the ovary of adult mice. The protective role of vitamin E (2 mg/kg body weight) along with their combination was also studied. Nickel and/or chromium to mice enhanced the levels of lipid peroxides in the ovary, which was accompanied by a significant decline in the levels of protein, glutathione, total ascorbic acid and activities of superoxide dismutase and catalase. Supplementation of vitamin E along with NiCl₂ + K₂Cr₂O₇ significantly lowered the levels of lipid peroxidation and enhanced the antioxidant status. Findings of the present study suggest that vitamin E exerts its protective effect against nickel and/or chromium induced toxicity by preventing lipid peroxidation and protecting antioxidant system in the mouse ovary.     

d-Amphetamine-induced hepatotoxicity: possible contribution of catecholamines and hyperthermia to the effect studied in isolated rat hepatocytes

Amphetamines are indirect-acting sympathomimetic drugs widely abused due to their physical and psychostimulating effects. However, the use of these drugs has been associated with numerous reports of hepatotoxicity. While glutathione depletion induced by amphetamines contributes to the exposure of hepatocytes to oxidative damage, other indirect effects attributed to amphetamines may have a role in cell injury. To examine this possibility, Wistar rats were used for plasma measurements of d-amphetamine and catecholamines (noradrenaline, adrenaline and dopamine) (15 min) after i.p. injection of d-amphetamine (5, 20 and 80 mg/kg). Freshly isolated rat hepatocytes were put into contact for 2 h with concentrations of d-amphetamine and catecholamines similar to those found in vivo. Since hyperthermia is a common consequence of acute amphetamine intake, the study using isolated hepatocytes was conducted at 37 °C and also at 41 °C in order to simulate high temperature levels. We found that hyperthermia was an important cause of cell toxicity: in vitro, a rise in incubation temperature from 37 to 41 °C causes oxidative stress in freshly isolated rat hepatocytes, as shown by a depletion of reduced glutathione (GSH; 23%), an increase of oxidized glutathione (GSSG; 157%), the induction of lipid peroxidation with 77% increase of thiobarbituric acid substances TBARS) and the consequent loss of cell viability (≤ 44%). Single treatment of isolated hepatocytes with catecholamines at 37 °C induced lipid peroxidation (29% increase of TBARS) but had no effect on glutathione or cell viability. Conversely, a single treatment with d-amphetamine induced glutathione depletion (≤ 24% depletion of GSH) with no effect on lipid peroxidation or cell viability. Also, d-amphetamine potentiated the induction by catecholamines of lipid peroxidation at 37 °C (≤ 48% increase of TBARS), while concomitant treatment of d-amphetamine and catecholamines potentiated cell death at 41 °C (≤ 56% of cell death) although no effect on viability was seen at 37 °C. It is concluded that the aforementioned modifications induced by d-amphetamine in vivo are cytotoxic to freshly isolated rat hepatocytes. Received: 30 October 1996 / Accepted: 13 January 1997     

In vitro haematotoxic effects of three methylated hydroxylamines

Hydroxylamine (HYAM, HONH₂) and some of its derivatives are known to cause erythrotoxic effects both in vitro and in vivo. Previous studies have shown that the primary in vitro effect of HYAM and O-ethyl hydroxylamine (OEH) is methaemoglobin formation, leading to liberation of free radicals which cause lipid peroxidation, enzyme inhibitions and glutathione depletion. By contrast, N-substituted N,O-dimethyl hydroxylamine (NODMH), primarily induces impairment of glucose 6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR). The oxidative potency of HYAM and the O-derivative was larger than the potency of the N,O-derivative. This seemed to indicate that attachment of an alkyl group to the nitrogen atom of hydroxylamine leads to decreased reactivity. To achieve a better understanding of the structure activity relationship for hydroxylamines three methylated derivatives were tested: N-methyl hydroxylamine (NMH), N-dimethyl hydroxylamine (NDMH) and O-methyl hydroxylamine (OMH). We were also interested in the erythrotoxic potency of OMH which recently entered industrial production. Methaemoglobin formation, high release of lipid peroxidation products, inhibition of NADPH methaemoglobin reductase and glutathione S-transferase (GST) and depletion of total glutathione (GT) were seen for OMH. The reducing enzymes G6PDH and GR were not impaired by OMH. These findings for OMH are consistent with the proposed mechanism for O-derivatives. Since both the effects caused by OMH and its potency are comparable to those of HYAM and OEH this indicates that possible occupational exposure to this compound may be approached similarly to HYAM and OEH. NMH only inhibited G6PDH and GR activity, which is fully in accord with the proposed mechanism for N-substituted derivatives of HYAM. However, NDMH a double N-substituted compound, caused a strikingly different scheme of reactivity: inhibition of G6PDH but not of GR, severe methaemoglobin formation, only little lipid peroxidation and some impairment of NADPH methaemoglobin reductase. This study confirms that O-derivatives of HYAM are potent haemoglobin oxidators, leading to other oxidative effects. The main effect was confirmed for single N-derivatives as inhibition of the two protective enzymes G6PDH and GR. However, the results for NDMH indicate that this simple classification of O-derivatives and N-derivatives has to be extended for double N-substituted compounds which give a mixture of effects. Received: 26 September 1996 / Accepted: 8 November 1996     

Elevated tissue Cr levels,increased plasma oxidative markers,and global hypomethylation of blood DNA in male Sprague‐Dawley rats exposed to potassium dichromate in drinking water

Hexavalent chromium [Cr (VI)] is prevalent in ground water in some areas, but evidence on the toxic effects of Cr (VI) via ingestion through drinking water remains insufficient. The aims of our study were to investigate the toxic effects of Cr (VI) through oral water ingestion on oxidative stress and DNA methylation. Thirty‐two Sprague‐Dawley rats were randomly divided into four groups, and exposed to porassium dichromate (K₂Cr₂O₇; 0, 30, 100, and 300 mg/L) in drinking water for 4 weeks. Mean body weight gain, mean water consumption, clinical chemistry determinations, and oxidative stress levels in plasma were measured. Global DNA methylation changes and DNA methylation status at the promoter of p16 gene were also detected. After 4 weeks, mild anemic effects and increased plasma malondialdehyde (MDA) levels occurred in rats exposed to 100 mg/L or 300 mg/L of Cr (VI). Plasma glutathione peroxidase (GSH‐Px) activity decreased in all exposed groups. Global DNA methylation levels were reduced in 100 mg/L and 300 mg/L exposure groups. However, DNA methylation status at the promoter of P16 gene remained unchanged in all K₂Cr₂O_7‐treated groups. The correlation analysis indicated that increased MDA levels were closely correlated to global DNA hypomethylation. Our results indicated that oral ingestion of Cr (VI) through drinking water caused not only oxidative stress in plasma, but also global DNA hypomethylation in blood cells from male rats, and a good correlation was found between increased MDA levels and reduced global DNA methylation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1080–1090, 2016.     

Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recently lipid peroxidation induced by OTA has been reported. OTA, a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid contamination of foodstuffs by toxigenic fungi, investigation is required for preventing the toxicity of OTA. An attempt to prevent its toxic effect, mainly the inhibition of protein synthesis, has been made using aspartame (l-aspartyl-l-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine. Protein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10–100 μM). After 24 h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC₅₀, was c.␣14.5 μM). Aspartame (A₁₉), at tenfold higher concentrations than OTA (100–1000 μM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24 h prior to the toxin (IC₅₀ 34 μM) than together (IC₅₀ 22 μM). As expected A₁₉(250 μM) prevented the OTA-induced leakage of certain enzymes, including lactate dehydrogenase, γ-glutamyl transferase, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25 μM)-treated cells. In order to investigate the effect of aspartame (A₁₉) on OTA-protein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in static diffusion cells with two compartments separated by a dialysis membrane. When A₁₉ (34 μM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240 μM) in the lower one, OTA binding was largely prevented (95–98%). When A19 (34 μM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240 μM), for the lowest concentration of OTA, the same efficiency was shown in preventing OTA binding, but at the two high concentrations A₁₉ seemed less efficient. Received: 9 July 1996 / Accepted: 1 October 1996     

Contribution to the mechanism of chromate nephrotoxicity in developing rats: EPR investigations

The effect of 2 mg and 1 mg Na₂Cr₂O₇ (Cr)/100 g body wt. on renal function was investigated in 10- and 55-day-old rats, respectively. These doses were followed by equal Cr concentrations in the renal tissue of both age groups. Confirming previous data we found lower nephrotoxicity in young than in adult rats. The concentration of glutathione (GSH) and the activity of glutathione reductase (GSSG reductase) in renal tissue of adult rats were diminished by buthionine sulfoximine (BSO) and lomustine (CCNU) administration, respectively. In these animals Cr nephrotoxicity was decreased significantly. Lower nephrotoxicity was accompanied by slower disappearance of Cr(VI) from renal tissue homogenate in vitro. The time course of Cr(VI) reduction demonstrated by the signal intensity of Cr(V), as recorded by electron spin resonance (EPR) spectroscopy in the supernatant of renal tissue homogenate, enabled us to follow the reduction of Cr(VI) to Cr(III) via Cr(V). Maximally reached Cr(V) concentrations, lowest in young rats, did not differ significantly in adult control and BSO and BSO + CCNU treated rats. Further reduction of Cr(V) to Cr(III) which appeared most rapidly in adult rats, was delayed by pretreatment with BSO and CCNU. From our results we concluded that (1) reduction of Cr(VI) was more related to the concentration of GSH than to the activity of GSSG reductase, (2) the formation of Cr-GSH-complexes without GSH oxidation seemed to be the first step of Cr(VI) metabolism, and (3) the stabilization of reactive Cr(V) by GSH seemed to be decisive for the preventive effect of BSO and CCNU as well as for age differences in chromate nephrotoxicity. Received: 21 May 1996/Accepted: 3 July 1996     

Toxic effects of chromium (VI) by maternal ingestion on liver function of female rats and their suckling pups

Potassium dichromate (K₂Cr₂O₇) is an environmental contaminant widely recognized as a carcinogen, mutagen, and teratogen toward humans and animals. This study investigated the effects of K₂Cr₂O₇ on the hepatic function of pregnant and lactating rats and their suckling pups. Experiments were carried out on female Wistar rats given 700 ppm of K₂Cr₂O₇ in their drinking water from the 14th day of pregnancy until day 14 after delivery. Hepatotoxicity was objectified by the significant increase in liver malondialdehyde content and a significant accumulation of chromium in this soft tissue. Moreover, exposure to K₂Cr₂O₇ induced a decrease of glutathione, nonprotein thiols, and vitamin C in the liver of mothers and their suckling pups. Alteration of the antioxidant system in the treated group was confirmed by the significant decline of antioxidant enzyme activities such as catalase, glutathione peroxidase, while liver superoxide dismutase activity increased in mothers and decreased in their offspring. It was found that K₂Cr₂O₇ induced liver damages as evidenced by the elevation of plasma aminotransferases, lactate dehydrogenase activities, and bilirubin levels. Impairment of the hepatic function corresponded histologically. Our investigation revealed hemorrhage, leukocytes infiltration cells, and necrosis, which were more pronounced in the hepatocytes of mothers than in those of their suckling pups. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

DNA binding activity of the mammalian translation elongation complex: recognition of chromium- and transplatin-damaged DNA

The elongation factor complex, EF-1H, serves an essential function in protein biosynthesis in eukaryotic cells, although the role of EF-1H in other physiological processes is unknown. In this report, we demonstrate that three components of EF-1H (EF-1β, EF-1δ, and EF-1γ) bind to DNA modified with chromium (Cr), a potent DNA-damaging agent and an established human carcinogen. The EF-1H complex also binds to transplatin modified DNA but not to cisplatin-modified DNA. These results demonstrate that the EF-1H complex has functional DNA binding activity and is capable of recognizing the distortions in DNA structure resulting from the covalent binding of Cr and transplatin to DNA. Received: 15 October 1996 / Accepted: 11 February 1997     

Influence of the route of administration and the chemical form (MnCl2, MnO2) on the absorption and cerebral distribution of manganese in rats

  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. Received: 2 May 1996 / Accepted: 21 August 1996     

Quantitation of α2-microglobulin after administration of structurally divergent chemical compounds

α₂-Microglobulin-induced nephropathy is a phenomenon which is exclusively found in adult male rats. Various chemicals are able to bind to α₂-microglobulin thus inhibiting its proteolytic degradation in lysosomes of the P2 segment of the rat nephron. The accumulation of this protein in `protein droplets' or `hyaline droplets' leads to necrosis, followed by regeneration which possibly later results in the formation of tumours. Here we report the development of a monoclonal antibody which is specific for α₂-microglobulin. It was utilized to measure α₂-microglobulin concentrations in plasma and tissues, and to stain α₂-microglobulin in fixed tissue slides. In two studies we administered to adult male Wistar rats two groups of compounds: (1) one group of structurally diverse compounds, which give an overview of chemical entities capable of inducing the accumulation of α₂-microglobulin; and (2) another group of structurally closely related compounds (i.e. substituted benzene derivatives) for the purpose of elucidating possible structure-activity relationships. The degree of α₂-microglobulin-induced nephropathy was determined by immunohistochemical staining of kidney sections. In addition, liver and kidney tissue and plasma concentrations of α₂-microglobulin were measured by competitive ELISA. Liver tissue and plasma concentrations of α₂-microglobulin were not found to be elevated whereas kidney tissue concentrations were higher than the controls. The increase over control values ranged␣from 154% (1,4-dichloromethyl-benzene) to 321% [α-methyl-4-(1-methylethyl)-cyclohexanemethanol]. Comparing structurally related benzene derivatives, the hyaline droplet accumulating (HDA) potential was found to depend both on the type of substituent and its position at the aromatic ring. In general HDA activity increased in the order benzene ≅ phenol ≅ alkylated phenols < halogenated phenols < halogenated␣benzenes. Further QSAR studies are needed to provide a theoretical base for these observations. Received: 18 September 1996 / Accepted: 18 December 1996     

Influence of subchronic administration of oestradiol, ethinyloestradiol and oestradiol sulphamate on bile flow, bile acid excretion, and liver and biliary glutathione status in rats

The cholestatic effect of the newly developed oestradiol sulphamate (J995) was compared with that of the natural oestrogen oestradiol (E) and of the cholestatic standard oestrogen ethinyloestradiol (EE). Female ovariectomized rats were orally treated for 7 days with oestrogen doses molar equivalent to 0.01, 0.1, 1, and 10 mg E/kg body wt. Bile flow, biliary bile acid and glutathione excretion as well as liver glutathione status were determined after bile duct cannulation under the influence of ketamine anaesthesia. For systemic oestrogen activity, the increase of uterine weight was measured. J995 showed the highest oestrogenic activity followed by EE and E. Impairment of bile flow and biliary glutathione excretion (GSH, GSSG) were found to be in the order E < J995 < EE. As all three oestrogens did not influence bile acid excretion, we postulate that mainly the bile acid-independent fraction of bile flow was inhibited, caused at least partly by impairment of canalicular glutathione transport. Based on the results of these studies, we conclude that a tenfold higher dose of J995 exhibited the same cholestatic effect as EE. Together with higher systemic oestrogenic activity, J995 may be used as a prodrug with reduced hepatic side-effects to replace EE in contraception strategies. Received: 25 October 1996 / Accepted: 27 February 1997     

Chromium(III) oxide nanoparticles induced remarkable oxidative stress and apoptosis on culture cells

Chromium(III) oxide (Cr₂O₃) is used for industrial applications such as catalysts and pigments. In the classical form, namely the fine particle, Cr₂O₃ is insoluble and chemically stable. It is classified as a low‐toxicity chromium compound. Recently, industrial application of nanoparticles (a new form composed of small particles with a diameter of ≤100 nm, in at least one dimension) has been increasing. Cellular effects induced by Cr₂O₃ nanoparticles are not known. To shed light upon this, the release of soluble chromium from Cr₂O₃ nano‐ and fine‐particles in culture medium was compared. Fine Cr₂O₃ particles were insoluble in the culture medium; on the contrary, Cr₂O₃ nanoparticles released soluble hexavalent chromium into the culture medium. Cr₂O₃ nanoparticles showed severe cytotoxicity. The effect of Cr₂O₃ nanoparticles on cell viability was higher than that of fine particles. Cr₂O₃ nanoparticles showed cytotoxicity equal to that of hexavalent chromium (K₂Cr₂O₇). Human lung carcinoma A549 cells and human keratinocyte HaCaT cells showed an increase in intracellular reactive oxygen species (ROS) level and activation of antioxidant defense systems on exposure to Cr₂O₃ nanoparticles. Exposure of Cr₂O₃ nanoparticles led to caspase‐3 activation, showing that the decrease in cell viability by exposure to Cr₂O₃ nanoparticles was caused by apoptosis. Cellular responses were stronger in the Cr₂O₃ nanoparticles‐exposed cells than in fine Cr₂O₃‐ and CrCl₃‐exposed cells. Cellular uptake of Cr₂O₃ particles were observed in nano‐ and fine‐particles. The cellular influence of the extracellular soluble trivalent chromium was lower than that of Cr₂O₃ nanoparticles. Cr₂O₃ nanoparticles showed cytotoxicity by hexavalent chromium released at outside and inside of cells. The cellular influences of Cr₂O₃ nanoparticles matched those of hexavalent chromium. In conclusion, Cr₂O₃ nanoparticles have a high cytotoxic potential. © 2011 Wiley Periodicals, Inc. Environ Toxicol 2013.     

Toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from Sambucus nigra : comparison with ricin

Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome-inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 °C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin. Received: 5 September 1996 / Accepted: 30 October 1996     

Low levels of cadmium chloride damage the corneal endothelium

The effect of cadmium chloride on the integrity of the endothelium of isolated bullfrog (Rana catesbeiana) corneas was examined by spectrophotometric analysis of corneal uptake of the vital stain Janus green and by scanning electron microscopy (SEM). The uptake of Janus green by the endothelium was dose related between 1.0 and 100.0 μM CdCl₂. The effect of cadmium was significantly attenuated by the calcium channel blocker SKF 96365 and was augmented by the calcium ionophore A23187, indicating that cadmium influx through calcium channels is an important determinant of its cellular effect. The effect of cadmium was not altered by changes in the external calcium concentration, indicating that the mechanism does not involve competitive inhibition by calcium. SEM demonstrated significant structural damage to the corneal endothelium exposed to cadmium chloride, including focal disruption and denuding of the apical endothelial membrane. Received: 11 October 1996 / Accepted: 12 February 1997     

Oral administration of potassium dichromate inhibits brush border membrane enzymes and alters anti-oxidant status of rat intestine

Potassium dichromate (K₂Cr₂O₇) is a soluble hexavalent chromium compound that is widely used in several industries. In the present work the effect of administration of K₂Cr₂O₇ on rat intestinal brush border membrane (BBM) enzymes and anti-oxidant system was studied. Rats were given a single oral dose of K₂Cr₂O₇ (100 mg/kg body weight) and sacrificed 6, 12, 24, 48 and 96 h after the treatment. Control animals were not given K₂Cr₂O₇. The administration of K₂Cr₂O₇ resulted in a reversible decline in the specific activities of several BBM enzymes. The decrease in the activities of these enzymes was due to changes in the maximum velocity while their affinities for the substrates remained unchanged. Lipid peroxidation increased while total SH groups decreased in K₂Cr₂O₇-treated rats as compared to controls indicating increased oxidative stress in the intestinal mucosa. The activities of superoxide dismutase and glutathione-S-transferase increased while those of catalase, glutathione reductase, thioredoxin reductase and glucose-6-phosphate dehydrogenase decreased. The maximum changes in all the parameters studied above were 24 h after administration of K₂Cr₂O₇ after which recovery took place, in most cases almost to control values after 96 h. These results show that oral administration of K₂Cr₂O₇ to decrease in the activities of BBM enzymes, increase in oxidative stress and alters the activities of anti-oxidant enzymes in rat intestine.     

Congener specific effects by polychlorinated biphenyls on catecholamine content and release in chromaffin cells

The effects of the non-planar polychlorinated biphenyl (PCB) congener 2,2′,4,4′-tetrachlorobiphenyl (2,4-TCB) and of the coplanar PCB congener 3,3′,4,4′-tetrachlorobiphenyl (3,4-TCB) were investigated on the catecholamine content and release from bovine adrenal chromaffin cells in culture. Each congener was tested at three concentrations (20, 50 and 100 μM) and two exposure periods (24 h and 5 days). Catecholamine release induced by K⁺-stimulation as well as catecholamine content of Triton X-100 treated cell cultures were examined using high-performance liquid chromatography (HPLC). 2,4-TCB showed dose- and time-dependent effects. 2,4-TCB at 100 μM reduced the K⁺-stimulated catecholamine release after 24 h of exposure. After 5 days of exposure, 2,4 TCB at 50 and 100 μM drastically reduced the K⁺-stimulated catecholamine release. 3,4-TCB even at a concentration of 100 μM over exposure of either 24 h or 5 days had no effects on the K⁺-stimulated secretion. When chromaffin cells, exposed to 2,4-TCB, were lysed with 0.5% Triton X-100, a dose- and time-dependent reduction of the catecholamine content appeared. The 3,4-TCB did not reduce the catecholamine content. Conversely there seemed to be a trend towards an increase in catecholamine content. Spontaneous release of catecholamines was strongly increased by the non-planar 2,4 TCB, while the coplanar 3,4 TCB showed no effects on this parameter. Furthermore, the effects of 2,4 TCB appeared to be reversible after replacing the highest concentration (100 μM) of the TCB-solution with culture-medium at the end of the 24-h exposure. Thus, K⁺-stimulated catecholamine release and the catecholamine content of bovine adrenal chromaffin cells was effectively reduced by the non-planar PCB congener whereas spontaneous catecholamine release was strongly increased. The coplanar PCB congener was ineffective at the same conditions. Received: 7 January 1997 / Accepted: 11 February 1997     

Implication of CYP 3A in the toxicity of cyclosporin G (CsG), cyclosporin A (CsA) and FK506 on rat hepatocytes in primary culture

FK506, cyclosporin A (CsA), and its structural analog cyclosporin G (CsG) are immunosuppressant drugs mainly metabolized by hepatic cytochrome P-450 3A (CYP 3A) oxygenase. FK506 metabolites exhibit greater toxicity than the parent drug, while CsA metabolites are far less toxic than CsA itself. The aim of our study was to compare the toxicity of CsG with CsA and FK506 as a function of CYP 3A induction. Hepatocytes from Wistar rats with or without dexamethasone (DEX) induction (200 mg/kg per day, p.o for 4 days) were used in primary culture. The DEX-inductive effect on CYP 3A was assessed by SDS-PAGE. After 6 h incubation with CsG, CsA or FK506 (5 to 200 μM), cell viability (expressed as IC₅₀), intracellular calcium content and apoptosis were evaluated. Concerning cytotoxicity, IC₅₀ values for CsG, CsA and FK506 were 75, 50 and 180 μM respectively in non-induced cultures, and 150, 120 and 25 μM in induced cultures. For intracellular calcium content, a dose-dependent increase was observed in all cultures. However this increase is more important for CsG and CsA in non-induced cultures (150%) compared to induced cultures (110%) at 150 μM. Conversely for FK506, this increase is greater in induced cultures (150%) than in non-induced cultures (127%). Estimation of the percentage of apoptotic cells shows similar variations. Our results show that the toxicity of the three drugs in rat hepatocytes is dependent on CYP 3A induction: increased for FK506, decreased for CsA and CsG. Moreover, with regard to the three tests used, the toxic effects of CsG are close to those of CsA, indicating that CsG metabolites are also less toxic than the parent drug. Received: 7 October 1996 / Accepted: 5 February 1997     

Lack of biliary excretion of Cd linked to an inherent defect of the canalicular isoform of multidrug resistance protein (cMrp) does not abnormally stimulate accumulation of Cd in the Eisai hyperbilirubinemic (EHB) rat liver

A new mutant, the Eisai hyperbilirubinemic (EHB) rat, shows no inherent expression of the canalicular isoform of the multidrug resistance protein (cMrp) in the liver. It has defective biliary secretion of organic anions such as bilirubin glucuronides, bromosulfophthalein (BSP), cysteinyl leukotrienes, glutathione (GSH) and bile acid sulfate and glucuronides. When rats were injected intravenously with CdCl₂, biliary excretion of Cd over 30 min was 0.28% and 0.004% of the total dose in Sprague-Dawley (SD) and EHB rats, respectively. Six SD rats and five EHB rats were fed a diet containing Cd. Bile Cd was detected at the level of 2 ng/20 min in SD rats, but not in EHB rats. There was no significant difference of hepatic Cd concentration between SD and EHB rats. Furthermore, there were no significant differences of renal and intestinal Cd, and hepatic and renal metallothionein (MT) concentrations between the SD and EHB groups. Biliary excretion of reduced-GSH for 20 min was 1.3 ± 0.3 mg and 3.6 ± 0.9 μg in SD and EHB rats, respectively. Our results suggest that hepatobiliary excretion of exogenous Cd is mediated mainly via carrier transport, including a cMrp or GSH carrier, but that the lack of the transport pathway does not contribute to abnormal accumulation of Cd in the liver. Received: 12 August 1996 / Accepted 7 November 1996     

Transplacental kinetics of lead in pregnant mini-pigs

This study examined the maternal and fetal blood kinetics of lead in pregnant, near term mini-pigs. A lead dose of 1 mg/kg was administered to the animals by i.v. injection as bolus. During the 5-h sampling period, the two-compartment maternal pharmacokinetics demonstrated a rapid phase T _1/2 of 8 min and a slower phase T_1/2 of 199 min. Lead reached the fetus with a time lag of 81 min. At 24 h after administration the ratio of fetal to maternal blood lead concentration seemed to become stable. When lead was injected directly into the fetal blood circulation, the decay of fetal blood lead fitted a one-compartment model. The disappearance half-life was 92 min. Lead can obviously accumulate in fetal liver; the lead level in fetal brain showed no detectable changes. This study confirmed that lead can also pass through the epitheliochorial placenta. Received: 1 August 1995 / Accepted: 21 August 1996