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Archives of Toxicology (v.72, #10)

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes by Patrick D. Lilly; Janice R. Thornton-Manning; Michael L. Gargas; Harvey J. Clewell; Melvin E. Andersen; Janice R. Thornton Manning (pp. 609-621).

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.

Keywords: Key words CYP2E1; Metabolic inhibition; Pharmacokinetic modeling; Chloroethylenes

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney by Ricarda Thier; Frederike A. Wiebel; Andreas Hinkel; Andreas Burger; Thomas Brüning; Konrad Morgenroth; Theodor Senge; Michael Wilhelm; Thomas G. Schulz (pp. 622-629).

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation␣between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/ rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

Keywords: Key words Glutathione transferase; GSTT1-1 ; Polymorphism ; Methyl chloride ; Dichloromethane ; 1; 2-Epoxy-3-(p-nitrophenoxy) propane

Characterization of CYP1A in hepatocytes of cynomolgus monkeys (Macaca fascicularis) and induction by different substituted polychlorinated biphenyls (PCBs) by A. S. A. M. Van Der Burght; A. P. Kreikamp; G. J. Horbach; W. Seinen; M. Van Den Berg (pp. 630-636).

Cynomolgus monkeys (Macaca fascicularis) have been used previously as a model to study effects on cytochrome P450 (CYP) regulation. Until now it has not been elucidated which CYP1A proteins are present in this primate species. The aim of this study was to characterize CYP1A in untreated hepatocytes of cynomolgus monkey using two specific CYP1A inhibitors (α -naphthoflavone and furafylline). The effect of different substituted polychlorinated biphenyls (PCBs) on CYP1A regulation was also studied in these hepatocytes. Small quantities of CYP1A2 have been identified in untreated hepatocytes. Northern blots showed the presence of a CYP1A mRNA in untreated hepatocytes, when hybridizations where performed with human CYP1A2 cDNA. Inhibitions with furafylline and α -naphthoflavone also suggested the presence of CYP1A2 properties. After induction with different PCBs, (probably) CYP1A1 mRNA and enzyme activity were induced in cynomolgus monkey hepatocytes. As expected, 2,3′,4,4′,5-PeCB (PCB no. 118), a mono-ortho substituted congener, was a potent CYP1A inducer but 2,2′,3,4,4′,5′,5′-HpCB (PCB no. 180), a di-ortho and 2,2′,3,4′,5,5′,6-HpCB (PCB no. 187), a tri-ortho substituted PCB, could induce CYP1A mRNA and enzyme activity in cynomolgus monkey hepatocytes as well.

Keywords: Key words Cytochrome P450 1A; Cynomolgus; monkey hepatocytes; Inhibitors; Polychlorinated biphenyls

Inhibition of prostaglandin-H-synthase by o -phenylphenol and its metabolites by Alexius Freyberger; Gisela H. Degen (pp. 637-644).

Chronic administration of o-phenylphenol (OPP) is known to induce urinary bladder tumours in the Fischer rat. The underlying toxic mechanism is poorly understood. Recently, arachidonic acid (ARA)-dependent, prostaglandin-H-synthase (PHS)-catalysed metabolic activation of the OPP metabolite phenylhydroquinone (PHQ) to a genotoxic species was suggested to be involved in OPP toxicity. To investigate this hypothesis in more detail, we have studied the effects of OPP and its metabolites on PHS. When microsomal PHS from ovine seminal vesicles (OSV) was used as enzyme source, both OPP, PHQ, and 2-phenyl-1,4-benzoquinone (PBQ) inhibited PHS-cyclooxygenase. The inhibitory potency was inversely related to the ARA concentration in the assay; at 7 μM ARA IC₅₀-values were: 13 μM (OPP), 17 μM (PHQ), and 190 μM (PBQ). In cells cultured from OSV, which express high PHS activity, 40 μM OPP almost completely suppressed prostaglandin formation. Studies with microsomal PHS demonstrated that PHQ was an excellent substrate for PHS-peroxidase; both ARA and hydrogen peroxide supported oxidation to PBQ. OPP was only a poor substrate for PHS, but inhibited the ARA-mediated and to a lesser extent also the hydrogen peroxide-mediated in␣vitro oxidation of PHQ. Moreover, PHQ at up to moderately cytotoxic concentrations (50 μM) did not induce micronuclei in OSV cell cultures. Taken together, our findings do not provide evidence for an ARA-dependent, PHS-catalysed formation of genotoxic species from PHQ. Moreover, it seems to be questionable whether such activation can effectively occur in vivo, since OPP and PHQ turned out to be efficient cyclooxygenase inhibitors, and high levels of OPP and PHQ were found at least in the urine of OPP-treated rats. On the other hand, inhibition of the formation of cytoprotective prostaglandins in the urogenital tract may play a crucial role in OPP-induced bladder carcinogenesis.

Keywords: Key wordso-Phenylphenol; Phenylhydroquinone; 2-Phenyl-1; 4-benzoquinone; Prostaglandin-; H-synthase; Rodent bladder carcinogenesis

Mutagenicity testing of organic extracts of diesel exhaust particles after spiking with polycyclic aromatic hydrocarbons (PAH) by Elisabet Bostrøm; Solveig Engen; Ingvar Eide (pp. 645-649).

In the present study, spiking was used as a strategy to evaluate the mutagenicity of individual compounds in a mixture. Mutagenicity of individual polycyclic aromatic hydrocarbons (PAH) was evaluated in an organic extract of diesel exhaust particles (DEP). The particles were extracted with dichloromethane (DCM). After replacing DCM with dimethylsulphoxide (DMSO), the extract was spiked with four individual PAH: benzo(a)pyrene, benzo(a)anthracene, pyrene and fluoroanthene. The PAH were added separately and in various combinations to the extract to determine the effects of each variable and to identify possible interactions between the individual PAH and between the PAH and the extract. The study was designed as a fractional factorial experiment with the five variables (the DEP extract and the four PAH), giving 16 (instead of 32) mixtures plus a triplicate centrepoint and background, i.e. a total of 20. The fractionated factorial design used in the present work supports a model with linear and interaction terms. The mixtures were tested for mutagenicity in the Ames assay using four strains of Salmonella typhimurium in the presence of rat liver xenobiotic enzymes (S9-mix). Projections to Latent Structures (PLS) was used to quantify the mutagenicity of each compound and possible interactions. The four individual PAH and the DEP extract acted additively in the Ames test with 10% S9-mix.

Keywords: Key words Combination toxicology; Complex mixtures; Diesel exhaust particles; Experimental design; Projections to latent structures

Murine strain differences and the effects of zinc on cadmium concentrations in tissues after acute cadmium exposure by Laura M. King; Mary B. Anderson; Suresh C. Sikka; William J. George (pp. 650-655).

The role of strain differences in cadmium tissue distribution was studied using sensitive (129/J) and resistant (A/J) mice. These murine strains have previously been shown to differ in their susceptibility to cadmium-induced testicular toxicity. Cadmium concentration was measured in testis, epididymis, seminal vesicle, liver, and kidney at 24 h after cadmium chloride exposure (4, 10, and 20 μmol/kg CdCl₂). The 129/J mice exhibited a significant increase in cadmium concentration in testis, epididymis, and seminal vesicle at all cadmium doses used, compared to A/J mice. However, cadmium concentrations in liver and kidney were not different between the strains, at any dose, indicating that cadmium uptake is similar in these organs at 24 h. These murine strains demonstrate similar hepatic and renal cadmium uptake but significantly different cadmium accumulation in the reproductive organs at 24 h. The mechanism of the protective effect of zinc on cadmium toxicity was studied by assessing the impact of zinc acetate (ZnAc) treatment on cadmium concentrations in 129/J mice after 24 h. Zinc pretreatment (250 μmol/kg ZnAc), given 24 h prior to 20 μmol/kg CdCl₂ administration, significantly decreased the amount of cadmium in the testis, epididymis, and seminal vesicle of 129/J mice, and significantly increased the cadmium content of the liver after 24 h. Cadmium levels in the kidney were unaffected at this time. Zinc pretreatment also prevented the cadmium-induced decrease in testicular sperm concentration and epididymal sperm motility seen in 129/J mice. These findings suggest that the differences in the two murine strains may be attributed partly to the differential accumulation of cadmium in murine gonads. This may be caused by strain differences in the specificity of cadmium transport mechanisms. The protective role of zinc in cadmium-induced testicular toxicity in the sensitive strain may be due to an interference in the cadmium uptake by susceptible reproductive organs.

Keywords: Key words Cadmium; Testis; Strain differences; Zinc

Regional selectivity to ochratoxin A, distribution and cytotoxicity in rat brain by A. Belmadani; G. Tramu; A. M. Betbeder; P. S. Steyn; E. E. Creppy (pp. 656-662).

Ochratoxin A (OTA) a chlorodihydro-isocoumarin linked through an amide bond to phenylalanine, is a mycotoxin found as a contaminant in foodstuffs and shown to be nephrotoxic, teratogenic, immunosuppressive, genotoxic, mutagenic and carcinogenic in rodents. Ochratoxin A is known to induce teratogenic effects in neonates (rats and mice) exposed in utero, characterised by microcephaly and modification of the brain levels of free amino acids. Since OTA has been found to accumulate in the brain according to the duration of exposure to doses in the range of natural contamination of feedstuffs, experiments were designed to determine more precisely the structural target of OTA in the brain. After intracerebral injection, OTA (403 ng/10 μl) was not found in the following parts of the brain : the frontal cortex (FC), striatum (ST), ventral mesencephalon (VM) and the cerebellum (CB) in contrast to the rest of the brain, probably due to the detection limit of 0.1 ng/g of tissue. However lactate dehydrogenase (LDH) was increased in extracellular space in the VM to a greater extent than in the rest of the brain, indicating that this structure could be one of the targets of OTA in the brain. Contents of free amino acids were morever similarly modified in the VM and in the rest of the brain. Male rats were given OTA (289 μg/kg per 24 h) by gastric intubation for 8 days and the main brain structures analysed for OTA content and cytotoxicity. OTA was found in the following structures in decreasing order: rest of the brain (50.3%), cerebellum (34.4%), VM (5.1%), striatum (3.3%) and hippocampus (2.9%) of the total OTA amount found in the brain, which represents 0.022% to 0.028% of the given dose. Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytotoxicity was observed in the rest of the brain. Similarly deoxyribonuclease (DNase) activity in relation to possible necrotic cells was increased in the VM and cerebellum. Altogether these results designated the ventral mesencephalon, hippocampus, striatum and cerebellum as the main OTA-targets in the brain of adult rats and excluded the rest of the brain.

Keywords: Key words Ochratoxin A; Rat brain; Stereotaxic injection; Regional selective cytotoxicity

Similar effects of cis-diamminedichloroplatinum(II) and cis-diammine-1, 1-cyclobutanedicarboxylatoplatinum(II) on sodium-coupled glucose uptake in renal brush-border membrane vesicles by Sophie Potdevin; Françoise Courjault-Gautier; Pierre Ripoche; Hervé J. Toutain (pp. 663-670).

cis-Diamminedichloroplatinum(II) (cDDP) has been shown to interfere with reabsorption processes in renal tubular epithelia, leading to polyuria, magnesium and sodium wasting and glucosuria. cDDP inhibits the Na⁺-coupled uptake of methyl-α-d-glucopyranoside (MGP) in renal proximal tubular cells in primary culture. cis-Diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (CBDCA) produces tubular injury qualitatively similar to that of cDDP with a reduced severity. CBDCA inhibits Na⁺-coupled MGP uptake in renal proximal tubular cells in primary culture at concentrations 20- to 30-times higher than those of cDDP. The Na⁺/glucose cotransport protein possesses sulphydryl groups (SH) essential for its activity. Platinum complexes have strong affinity for SH groups. We compared the direct effects of cDDP (0.04–1.0 mM) and CBDCA (1–30 mM) on Na⁺-coupled MGP uptake in rabbit renal brush-border membrane (BBM) vesicles. cDDP and CBDCA inhibited Na⁺-coupled MGP uptake in a concentration-dependent manner, mainly through a decrease in V _max of the cotransport protein. These effects were associated with platinum binding to BBM and decreases in protein-bound SH groups. CBDCA altered Na⁺-coupled MGP uptake at concentrations 30-times higher than those of cDDP. When BBM vesicles were preincubated with cDDP or CBDCA, diethyldithiocarbamate (an antidote against cDDP-induced nephrotoxicity) partly restored Na⁺-coupled MGP uptake and reduced the amount of platinum bound to BBM, but did not restore protein-bound SH groups. These findings strongly suggest that the inhibition of Na⁺-coupled MGP uptake by cDDP and CBDCA is mainly mediated by direct chemical binding of platinum to essential SH groups of the cotransport protein but may also involve other nucleophilic groups, such as the SCH₃group of methionine residues.

Keywords: Key words Kidney; Sodium/glucose cotransport; Cisplatin; Carboplatin; Sulphydryl groups

Quantitative analysis of O-isopropyl methylphosphonic acid in serum samples of Japanese citizens allegedly exposed to sarin: estimation of internal dosage by Daan Noort; Albert G. Hulst; Dominique H. J. M. Platenburg; Martine Polhuijs; Hendrik P. Benschop (pp. 671-675).

A convenient and rapid micro-anion exchange liquid chromatography (LC) tandem electrospray mass spectrometry (MS) procedure was developed for quantitative analysis in serum of O-isopropyl methylphosphonic acid (IMPA), the hydrolysis product of the nerve agent sarin. The mass spectrometric procedure involves negative or positive ion electrospray ionization and multiple reaction monitoring (MRM) detection. The method could be successfully applied to the analysis of serum samples from victims of the Tokyo subway attack and of an earlier incident at Matsumoto, Japan. IMPA levels ranging from 2 to 135 ng/ml were found. High levels of IMPA appear to correlate with low levels of residual butyrylcholinesterase activity in the samples and vice versa. Based on our analyses, the internal and exposure doses of the victims were estimated. In several cases, the doses appeared to be substantially higher than the assumed lethal doses in man.

Keywords: Key words Chemical warfare agents; LC tandem MS; Multiple reaction monitoring; O-isopropyl methylphosphonic acid; Sarin

Cloning and sequence of guinea pig interleukin 2 (IL-2) by Masahiro Takeyoshi; Hiroyuki Iwata; Takeshi Inoue (pp. 676-678).

Interleukin 2 (IL-2) is a T-cell proliferation factor released from TH0- and TH1-type helper T-cells and is an essential cytokine for certain immune responses. We reported here cloning and sequence of IL-2 cDNA in guinea pigs, which have been used for a long time in various immunological experiments and in vivo screening tests for skin sensitization potential of chemicals. Consequently, a cDNA clone was obtained encoding guinea pig IL-2 of 520 bp in length, which contained a complete open reading frame. Alignment of the amino acid sequence with human IL-2 indicates that guinea pig IL-2 is composed of 20 amino acids (aa) of a signal peptide and 132 aa of a mature peptide with a predicted molecular weight of 15 133. Guinea pig IL-2 has an amino acid homology of 62% with human IL-2, 52% with murine IL-2, and 55% with rat IL-2. In addition, guinea pig IL-2 has a possible N-linked glycosylation site as seen in bovine and porcine IL-2.

Keywords: Key words Cloning; Guinea pig; Interleukin 2 (IL-2)

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Archives of Toxicology (v.72, #10)