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Archives of Toxicology (v.74, #12)
No Title by P. Ulrich; J. Streich; W. Suter (pp. 733-744).
We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo)allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.
Keywords: Contact allergy Contact photoallergy Irritation Photoirritation Local lymph node assay (LLNA)
No Title by Berend T. Leussink; Anja Slikkerveer; Marc R.W. Engelbrecht; Gijsbert B. van der Voet; Etienne J. Nouwen; Emile de Heer; Marc E. de Broe; Frederik A. de Wolff; Jan A. Bruijn (pp. 745-754).
Overdosing of colloidal bismuth subcitrate (CBS), used to treat peptic ulcers and Helicobacter pylori infections, has been reported to result in serious, though reversible, nephrotoxicity in humans. However, little is known about the nature of the renal damage induced by bismuth (Bi), and no well-described experimental model exists. Single large oral CBS doses (0.75, 1.5, and 3.0 mmol Bi/kg) were administered to three groups of 20 female Wistar rats. A control group (n=20) received only the vehicle. Standard kidney function parameters, urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and the Bi content were monitored in blood, urine, liver, and kidneys for 14 days. A dose of 3.0 mmol Bi/kg, 100 times the daily therapeutic dose, caused kidney damage within 6 h as detected by proteinuria, glucosuria, and elevated plasma urea and plasma creatinine levels. The kidneys of all animals, except two that died, recovered functionally within 10 days. At a dose of 1.5 mmol Bi/kg, clinical parameters changed less and normalized within 48 h, whereas a dose of 0.75 mmol Bi/kg induced no changes. Histological evaluation revealed that the S3 tubular segment necrotized first with additional necrotization of the S1/S2 segment when more Bi was absorbed. The lesions were accompanied by interstitial infiltrates of CD45+ leukocytes. In summary, we developed a rat model for Bi-induced reversible nephropathy. A large single oral overdose of CBS administered to Wistar rats led to damage to the proximal tubule, especially in the last segment.
Keywords: Bismuth Nephrotoxicity Nephropathy Rat Proximal tubule
No Title by Tetsuo Nomiyama; Hiroshi Nakashima; Yuri Sano; Li Ling Chen; Shigeru Tanaka; Hiroyuki Miyauchi; Tsuneyuki Yamauchi; Haruhiko Sakurai; Kazuyuki Omae (pp. 755-759).
The aim of this study was to clarify whether phenotypic variation exists when subjects with different genotypes of cytochrome P450 2E1 (CYP2E1) are exposed to N,N-dimethylformamide (DMF). The genotypes of CYP2E1 were confirmed in 123 healthy male volunteer subjects. Of the 123 subjects, the numbers of c1 homozygotes, c2 heterozygotes, and c2 homozygotes were 77, 45, and 1, respectively. Seven of the c1 homozygotes, five of the c2 heterozygotes, and the one c2 homozygote (mean age: 22.7 years, range: 20–27 years) were exposed to DMF vapor twice, once via the skin and once via the lung, for a total of 8 h per subject at a concentration below 10 ppm, the occupational exposure limit recommended by the Japan Society for Occupational Health, the American Conference of Governmental and Industrial Hygienists, and Deutsche Forschungsgemeinschaft, at 27°C and 44% relative humidity. Exposure levels were 6.2±1.0 ppm in dermal exposure and 7.1±1.0 ppm in inhalation exposure. Urine samples were collected until 72 h after exposure. The half-lives of urinary N-methylformamide (NMF) were obtained as the phenotype. The average urinary NMF half-lives of the c1 homozygotes, the c2 heterozygotes, and the c2 homozygote were 3.86±1.90, 4.38±1.53, and 4.2 h after dermal exposure, and 1.58±0.42, 1.84±0.61, and 3.2 h after respiratory exposure. The NMF half-lives of the c1 homozygotes were not significantly different from those of the c2 heterozygotes, and there were no differences between the NMF half-lives on the subjects with and without the c2 allele. Even though the data were obtained from only one c2 homozygote, it is noteworthy that the NMF half-life of this subject was slightly less than that of the c1 homozygotes after respiratory exposure.
Keywords: Cytochrome P450 2E1 Genetic polymorphism Genotype Phenotype N,N-Dimethylformamide
No Title by Michael Müller; Michael Voss; Christian Heise; Thomas Schulz; Jürgen Bünger; Ernst Hallier (pp. 760-767).
Glutathione-S-transferase T1 (GSTT1-1) is a major isoenzyme for the biotransformation of halomethanes. The enzyme activity is located, among other places, in human liver and erythrocytes and is subject to a genetic polymorphism. Metabolism of the halomethanes via GSTT1-1 yields S-methylglutathione (MeSG). A new HPLC assay for the enzymatic formation of MeSG was developed. The glutathione conjugate was derivatized with 9-fluorenylmethyl chloroformate, followed by reverse-phase HPLC with gradient elution and fluorescence detection. The limit of detection was as low as about 39 pmol MeSG on-column. Including derivatization and HPLC analysis, samples could be run at 42-min intervals, thus enabling a high sample throughput. The entire method was validated for analyte recovery (78.2%) and for variations in detector response with replicated injections (11.8%) and with analyses on each of 11 consecutive days (15.2%) with erythrocyte lysate incubations as the matrix. The time-, protein-, and substrate-dependences of the enzymatic catalysis with the model substrates methyl bromide (MeBr) and methyl chloride (MeCl) were studied. Due to its strong electrophilic character, MeBr caused a high level of spontaneous MeSG formation from glutathione in a protein-free medium and a substrate-trapping side reaction in the presence of proteins. Therefore, enzymatic MeSG formation rates may only be determined with MeBr concentrations of at least 3000 ppm in the presence of limited amounts of protein (e.g. 100 µl erythrocyte lysate). In contrast, MeCl showed a lower alkylating potential allowing enzymatic catalysis to be the dominant reaction in incubations with 10,000 ppm MeCl and 2 ml erythrocyte lysate.
Keywords: Glutathione-S-transferase T1 Methyl bromide Methyl chloride S-Methylglutathione HPLC/fluorescence detection analysis
No Title by Abraham Nyska; Liat Lomnitski; Robert Maronpot; Cindy Moomaw; Berta Brodsky; Amnon Sintov; Uri Wormser (pp. 768-774).
In a previous study we demonstrated the protective effect of topical iodine as postexposure treatment for sulfur mustard (SM) application. The iodine treatment results in significantly reduced inflammation and necrosis and increased epidermal hyperplasia. The expression and localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in paraffin-embedded skin samples from that study were evaluated in the present investigation. We compared the immunoreactivity of iNOS and COX-2 using five samples from each of the following four test sites: untreated control sites, SM-exposed sites, sites treated with iodine mixture 15 min after SM exposure, and sites treated with iodine 30 min after SM exposure. All animals were killed 2 days after irritant exposure. iNOS immunoreactivity was present only in skin sites exposed to SM without iodine treatment. The ulcerated skin was covered with a relatively thick band of exudate composed of iNOS-immunostained polymorphonuclear cells and macrophages. In untreated skin, COX-2 immunostaining was limited to the thin suprabasal epidermal layer. In SM-exposed skin, induction of COX-2 was noted in inflammatory cells located close to the site of epidermal injury. In skin sites treated with iodine 15 or 30 min after SM exposure, the regenerating hyperplastic epithelium showed moderate cytoplasmic staining localized to the epithelium overlying the basal layer. This pattern of staining was also present in the nearby dermal fibroblasts. Thus, in contrast to the skin samples exposed to SM without iodine treatment, the epidermal layer expressing immunohistochemical positivity for COX-2 was thicker and corresponded to the epidermal hyperplasia noted in samples treated with iodine. It is well documented that prostaglandins (PGs) promote epidermal proliferation, thereby contributing to the repair of injured skin. That the induction of the COX-2 shown in our study may also play a role in the healing process is indicated by the present evidence. The results suggest that nitric oxide radicals (NO·) are involved in mediating the damage induced by the SM and that iodine-related reduction in acute epidermal inflammation is associated with reduced iNOS expression.
Keywords: Sulfur mustard Skin COX-2 iNOS
No Title by Shuichi Hara; Toshiji Mukai; Kunihiko Kurosaki; Fumi Kuriiwa; Takeshi Yanase; Sadao Kano; Takahiko Endo (pp. 775-782).
Shaking behavior, so-called wet dog shakes (WDS), in rats is characteristic behavior indicating morphine abstinence in morphine-dependence and central excitation in relation to seizures elicited by chemicals or electrical stimulation. We have found that paraquat (PQ), a nonselective herbicide, administered systemically to rats induces WDS in a dose-dependent manner. PQ-induced WDS are suppressed by nitric oxide (NO) synthase (NOS) inhibitors, but this suppression is not reversed by an NO precursor, L-arginine (L-Arg). The present study was performed to determine whether the NO system is associated with PQ-induced WDS in rats. A time-course study on the frequency of WDS for each 30-min period up to 120 min after PQ administration (70 mg/kg, s.c.) revealed that significant induction of WDS occurred during the first and second 30-min periods, that is within 60 min of PQ administration. A nonselective NOS inhibitor, Nω-nitro-L-arginine (L-NA; 30 mg/kg, i.p.), reduced the frequency of the PQ-induced WDS during both of these periods, but the reduced frequency was not reversed by L-Arg (500 mg/kg, i.p.) in either period. Significant induction of WDS occurred when PQ (50 nmol) was administered directly into the ventral or dorsal hippocampus, but not when administered into the amygdala or the caudate putamen, indicating that the hippocampus plays an important role in PQ-induced WDS. The WDS after the administration of PQ into the dorsal hippocampus was significantly suppressed by pretreatment with L-NA (30 mg/kg, i.p.). The extracellular levels of nitrite (NO2 –) and nitrate (NO3 –), the oxidative products of NO, in the dorsal hippocampus determined by in vivo microdialysis, were stimulated after systemic PQ administration (70 mg/kg, s.c.) in urethane-anesthetized rats. The increases in extracellular NO2 – and NO3 – were inhibited by L-NA (30 mg/kg, i.p.), and this inhibition was partly reversed by L-Arg (500 mg/kg, i.p.). The increases in extracellular NO2 – and NO3 – in the dorsal hippocampus appeared 60 min after PQ administration, when the WDS had occurred and disappeared. These findings suggest that NO production in the hippocampus plays a minor role in PQ-induced WDS in rats and that the suppression of PQ-induced WDS by NOS inhibitors might be mediated though complex mechanisms in the brain.
Keywords: Paraquat Wet dog shakes Nitric oxide Nω-Nitro-L-arginine Rat
No Title by Edward A. Lock; Andrew Gyte; Stephen Duffell; Ian Wyatt (pp. 783-788).
L-2-Chloropropionic acid (L-2-CPA) selectively damages the cerebellum in adult rats. The rat cerebellum continues to develop postnatally during the first 4 weeks of life. In this study we examined the neurotoxic effect on rats of increasing postnatal age. Daily oral dosing of rats aged 56 days with 250 mg/kg per day of L-2-CPA for 3 days produced necrosis to neurons in the cerebellar granule cell layer and to neurons in the medial/ventral region of the habenular nucleus. Rats aged 22 days were resistant to the cerebellar toxicity while rats aged 32 days and older were sensitive. A single large oral dose of 500 or 750 mg/kg L-2-CPA produced no clinical signs of neurotoxicity or lesions in the cerebellum 48 h after dosing in 22-day-old rats. Daily dosing of 22-day-old rats at 250 mg/kg per day L-2-CPA for 10 days also produced no signs of neurotoxicity or reduction in body weight gain, although histological examination of the brain revealed slight neuronal cell necrosis in the granule cell layer of the cerebellum with a minimal effect in the medial/ventral region of the habenular nucleus. In contrast, daily dosing of rats aged 32, 38, 48 and 58 days with 250 mg/kg per day of L-2-CPA for 3 days produced clear signs of neurotoxicity which were associated with reduced body weight gain and loss of hindlimb function. In these rats there was clear evidence of neuronal cell loss in the cerebellar granule cell layer and medial/ventral region of the habenular nucleus. This study showed that the postnatal developing cerebellum is resistant to L-CPA-induced injury in rats up to 25 days of age, but becomes vulnerable to the toxicity by 32 days of age. The basis for the resistance of the developing cerebellum to L-CPA is discussed.
Keywords: Postnatal toxicity of L-2-chloropropionic acid in the rat Cerebellar granule cell necrosis Necrosis to the habenular nucleus
No Title by Márcia Carvalho; Félix Carvalho; Maria Lourdes Bastos (pp. 789-793).
The consumption of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) may cause hepatocellular damage in humans, a toxic effect that has been increasing in frequency in the last few years, although the underlying mechanisms are still unknown. The metabolism of MDMA involves the production of reactive metabolites which form adducts with intracellular nucleophilic sites, as is the case with glutathione (GSH). Also, MDMA administration elicits hyperthermia, a potentially deleterious condition that may aggravate its direct toxic effects. Thus, the objective of this study was to evaluate the extent of MDMA-induced depletion of GSH, induction of lipid peroxidation and loss of cell viability in freshly isolated mouse hepatocytes under normothermic conditions (37°C) and to compare the results with the effects obtained under hyperthermic conditions (41°C). By itself, hyperthermia was an important cause of cell toxicity. A rise in incubation temperature from 37°C to 41°C caused oxidative stress in freshly isolated mouse hepatocytes, reflected as a time-dependent induction of lipid peroxidation and consequent loss of cell viability (up to 40–45%), although the variations in GSH and GSSG levels were similar to those under normothermic conditions. MDMA (100, 200, 400, 800 and 1600 µM) induced a concentration- and time-dependent GSH depletion at 37°C but had a negligible effect on lipid peroxidation and cell viability at this temperature. It is particularly noteworthy that hyperthermia (41°C) potentiated MDMA-induced depletion of GSH, production of lipid peroxidation and loss of cell viability (up to 90–100%). It is therefore concluded that hyperthermia potentiates MDMA-induced toxicity in freshly isolated mouse hepatocytes.
Keywords: MDMA Hyperthermia Freshly isolated mouse hepatocytes Hepatotoxicity
No Title by Bożena Chłopkiewicz (pp. 794-798).
The influence of metabolic activation on the genotoxic activity of the antihypertensive drugs hydralazine and dihydralazine was investigated. An in vitro micronucleus test for estimating the genotoxic activity of these drugs was used. The results obtained indicated that hydralazine and dihydralazine induce micronuclei formation in L929 cells. When L929 cell cultures were treated with drugs together with liver membrane fraction (S9 fraction) from polychlorinated biphenyl (Aroclor 1254) induced rat liver, the number of micronucleated cells decrease, however, almost to the level found in control cultures. The experiments with modified S9 mix allow the conclusion that the antioxidant enzymes catalase and superoxide dismutase present in S9 liver fraction play a role in the protection of cells from the genotoxic action of hydralazine and dihydralazine.
Keywords: Hydralazine Dihydralazine Genotoxicity Metabolic activation Micronucleus test