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Archives of Toxicology (v.78, #6)

Simultaneous determination of hydrogen cyanide and volatile aliphatic nitriles by headspace gas chromatography, and its application to an in vivo study of the metabolism of acrylonitrile in the rat by Miyako Shibata; Kouichi Inoue; Yoshihiro Yoshimura; Hiroyuki Nakazawa; Yasuo Seto (pp. 301-305).

A method for the simultaneous determination of hydrogen cyanide (HCN) and aliphatic nitriles using manual headspace (HS) gas chromatography (GC) with a capillary porous polymer column GS-Q and a nitrogen–phosphorus detector is described. With a HS incubation at 50°C for 30 min and a GC temperature at 180°C, HCN and volatile nitriles [acetonitrile, acrylonitrile (VCN), propionitrile, isobutyronitrile] were well separated and could be detected within 7 min with a detection limit of 0.7–2.4 ng/ml in blood samples. The HS-GC method was used in an in vivo study of VCN metabolism. VCN was administered orally (at nearly one-half its LD₅₀) to rats, and heart blood and urine were sampled. Blood concentrations of HCN and VCN were measured by HS-GC, and plasma and urinary thiocyanate concentrations were measured by the König colorimetric method. Blood levels of HCN and VCN peaked 1.5 h after VCN administration, at which time the cyanide level (about 0.7 μg/ml) is close to the fatal level. HCN levels were observed to be at almost background levels at 10 h, although 50 ng/ml VCN was still detectable. The plasma thiocyanate level increased, reaching a peak (about 30 μg/ml) at 5 h. The cumulative urinary thiocyanate amount gradually increased, and at 10 h more than 1 mg thiocyanate was excreted into the urine. It is therefore possible to clarify the cause of cyanide poisoning using HS-GC analysis, when someone has taken volatile nitriles.

Keywords: Headspace gas chromatography; Cyanide; Nitrile; In vivo metabolism

Genetic polymorphisms of CYP1A1 in a Korean population by Duk Woong Park; Bohwan Jin; Dongdeuk Jang; Kihwa Yang; Jung-Duck Park; Yong-Soon Lee; Doug-Young Ryu (pp. 306-308).

Genetic polymorphisms in the coding exons of the CYP1A1 gene were analyzed in 100 Koreans. Three types of CYP1A1 polymorphisms, specifically G134A, G184C and A2455G, were identified with allelic frequencies of 18, 3, and 16%, respectively, and no linkage was observed among them. The novel G184C polymorphism identified in this study was associated with the mutation of an alanine residue at position 62 to proline. Other earlier-reported polymorphisms in the coding region of CYP1A1 were not detected.

Keywords: CYP1A1 ; Genetic polymorphism; Human

Role of the aryl hydrocarbon receptor and Cyp1b1 in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin by Kei Takemoto; Miki Nakajima; Yuto Fujiki; Miki Katoh; Frank J. Gonzalez; Tsuyoshi Yokoi (pp. 309-315).

The role of aryl hydrocarbon receptor (AhR) and cytochrome P450 (Cyp) 1 family in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in vivo. Immature (21 days old) AhR, Cyp1a2, or Cyp1b1 knockout (−/−) mice were treated intraperitoneally with estradiol (E2, 20 ng/mouse per day, for 14 consecutive days) and/or TCDD (200 ng/mouse per day, on days 7, 9, 11, and 13). Uterine wet weight and uterine peroxidase activity (UPA) were measured as markers of estrogen responsiveness. UPA was a better marker of estrogen responsiveness than the uterine wet weight. In AhR wild-type (+/+) mice, UPA (208.1±81.6 units/g tissue) was increased by the administration of E2 (to 297.2±178.7 units/g). The administration of TCDD significantly (p<0.01) decreased the UPA (10.5±3.4 units/g) compared with that in the control mice. Co-administration of TCDD with E2 also significantly (p<0.05) decreased the UPA (18.8±19.9 units/g) compared with that in E2-treated mice. In AhR(−/−) mice, UPA (162.9±146.7 units/g) was significantly (p<0.01) increased by the administration of E2 (486.8±108.2 units/g). In contrast to the results in AhR(+/+) mice, UPA was not affected by the administration of TCDD (51.8±70.6 units/g) compared with control, and co-administration of TCDD with E2 (545.8±189.4 units/g) compared with that in E2-treated mice. In Cyp1a2/1b1(+/+) mice, UPA was significantly (p<0.05) increased by the administration of E2 (70.0±36.4 units/g). Co-administration of TCDD with E2 significantly (p<0.05) decreased the UPA (29.6±22.2 units/g) compared with that in E2-treated mice. In Cyp1a2(−/−) mice, co-administration of TCDD with E2 significantly (p<0.01) decreased the UPA (6.8±5.1 units/g) compared with that in E2-treated mice. In Cyp1b1(−/−) mice, UPA (5.5±8.1 units/g) was significantly (p<0.05) increased by the administration of E2 (56.6±34.1 units/g). In contrast to the results in Cyp1a2/1b1(+/+) mice or Cyp1a2(−/−) mice, UPA was not affected by the co-administration of TCDD and E2 (52.6±30.1 units/g) compared with that in E2-treated mice. This is the first demonstration that Cyp1b1 as well as AhR is involved in the antiestrogenic effects of TCDD.

Keywords: Cyp1b1; Aryl hydrocaron receptor; Estrogen receptor; 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD); Antiestrogenic activity

Aroclor 1254 inhibits tryptophan hydroxylase activity in rat brain by Izhar A. Khan; Peter Thomas (pp. 316-320).

Previous studies have shown that oral exposure of rats to polychlorinated biphenyls (PCBs) results in reduced 5-hydroxytryptamine (5-HT) concentrations in certain brain regions. In the present study, we investigated whether the PCB mixture Aroclor 1254 (0.33 mg/g body weight as a single oral dose) can inhibit the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, and reduce 5-HT concentrations in selected brain areas. In two separate experiments, Aroclor 1254 exposure consistently reduced TPH activity in the brainstem (7.2 and 8.7%), frontal cortex (17.4 and 14.8%), and hypothalamus (10.7 and 9.4%) without altering the rats’ food intake or growth. Moreover, Aroclor 1254 accumulation in the frontal cortex demonstrated a negative correlation with TPH activity (correlation coefficient −0.82). In addition, 5-HT concentrations decreased in the brainstem and frontal cortex after Aroclor 1254 exposure by 9.1 and 19.7%, respectively. These results suggest that the Aroclor 1254-induced decreases in 5-HT concentrations in certain areas of the rat brain are due to inhibition of TPH activity, similar to our recent observations in Atlantic croaker, and that TPH is one of the targets of PCB neurotoxicity in both fish and mammals.

Keywords: Polychlorinated biphenyls; Tryptophan hydroxylase; 5-Hydroxytryptamine; Neurotoxicity; Rats

The hepatoprotective effect of putrescine against cadmium-induced acute liver injury by Konstantinos N. Tzirogiannis; Georgios I. Panoutsopoulos; Maria D. Demonakou; George K. Papadimas; Vasiliki G. Kondyli; Kalliopi T. Kourentzi; Rosa I. Hereti; Michael G. Mykoniatis (pp. 321-329).

The hepatoprotective effect of putrescine against cadmium liver injury was investigated. Male Wistar rats were injected with a dose of cadmium (6.5 mg CdCl₂/kg bodyweight, intraperitoneally). Normal saline (group I) or putrescine (300 µmol/kg bodyweight; group II) were injected 2, 5 and 8 h later. A number of animals of both groups were killed 0, 12, 16, 24, 48 or 60 h after cadmium intoxication. Liver tissue was histologically assessed for necrosis, apoptosis, peliosis, mitoses, and inflammatory infiltration. Apoptosis was also quantified by the TUNEL assay for hepatocytes and nonparenchymal liver cells. The discrimination between hepatic cell subpopulations was achieved histochemically. The mitotic index in hematoxylin–eosin-stained sections and by the immunochemical detection of Ki67 nuclear antigen, ³H-thymidine incorporation into hepatic DNA, and hepatic thymidine kinase activity were all used as indices of liver regeneration. Both hepatocyte apoptosis and liver necrosis evolved in a biphasic temporal pattern. Nonparenchymal cell apoptosis and peliosis hepatis evolved in a monophasic pattern and were correlated closely. Putrescine administration totally reversed liver necrosis and hepatocyte apoptosis. The time profile of nonparenchymal apoptosis was altered and peliosis hepatis was also totally attenuated. In conclusion, putrescine protected hepatocytes and modulated the mechanism of cadmium-induced acute hepatotoxicity.

Keywords: Apoptosis; Cadmium; Hepatotoxicity; Necrosis; Peliosis; Putrescine

Effects of the carbamates fenoxycarb, propamocarb and propoxur on energy supply, glucose utilization and SH-groups in neurons by Gabriele Schmuck; Florin Mihail (pp. 330-337).

Carbamates belonged to an older insecticide group, with propoxur being representative of this group. However, today carbamates with hormonal effects on insects, like fenoxycarb, or with fungicide properties, like propamocarb, are also used. The goal was a comparison of three structurally and functional different carbamates with a possibly common toxicological mechanism. Primary neuronal cell cultures of the rat are a well established model to identify neurotoxic compounds like n-hexane or acrylamide. In this cell culture model endpoints such as viability, energy supply, glucose consumption, glutathione (GSH) levels and cytoskeleton elements were determined. Added to cultured rat cortical neurons for 1 week, fenoxycarb, propamocarb and propoxur considerably decreased ATP levels, mitochondrial membrane potential and glucose consumption. Besides this, fenoxycarb and propamocarb had an impact on neurofilaments. After recovery for 1 week, propoxur also showed effects on neurofilaments, whereas with the other carbamates no tendency for a recovery was seen. These effects were prevented completely by pyruvate for propoxur and propamocarb, and partly so for fenoxycarb. In contrast to the main experimental design, GSH was determined after 1-h treatment with the test substances. Surprisingly, the compounds had only slight or no effect on the GSH level within this time. Further mechanistic studies indicated that carbamates primarily interacted with SH-groups, most likely by interfering with glycolysis and the construction of fibrillary proteins like neurofilaments. The prevention by pyruvate and acetylcysteine pointed to these biochemical endpoints.

Keywords: Glycolysis; Cellular energy supply; Neurofilaments; Glutathione

Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase by S. Herkenhoff; L. Szinicz; V. K. Rastogi; T.-C. Cheng; J. J. DeFrank; F. Worek (pp. 338-343).

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

Keywords: Acetylcholinesterase; Organophosphate; Obidoxime; Phosphonyloxime; Organophosphorus hydrolase

Heat shock protein 70 in the rat nasal cavity: localisation and response to hyperthermia by Sharon A. Simpson; David J. Alexander; Celia J. Reed (pp. 344-350).

Heat shock proteins (HSPs) are a group of proteins that are rapidly induced in response to physiological stress, including hyperthermia and exposure to toxicants. Thus they may provide a useful index of toxicity in in vitro systems for screening for toxicity. We have recently developed a rat nasal explant system for investigating upper respiratory tract toxicity, and the aims of this study were to localise HSP70 within the rat nasal cavity and to characterise its response to hyperthermia. Constitutively, HSP70 was found to be predominantly localised to the sustentacular cells, basal cells and Bowman’s glands of the olfactory epithelium (OE), with the most intense immunohistochemical staining at levels 3 and 4 of the posterior of the rat nasal cavity. Ethmoturbinates (ETs) and liver slices were exposed to heat shock (37° and 43°C, respectively) for 45 min and then returned to normal culture temperatures (31° and 37°C, respectively) for 24 h. In ETs, HSP72 was maximally induced 4-fold at 4 h after heat shock, and levels then returned to those of control tissue. ATP concentrations were markedly decreased up to 4 h after heat shock and then returned to control levels. In contrast, HSP72 levels in liver slices increased and ATP levels decreased steadily throughout the 24 h culture period. ETs were also able to withstand a 45-min heat shock at 43°C, that is 12°C above normal culture temperature. Incubation of ETs with cycloheximide prior to heat shock reduced the ability of the OE to recover from heat shock at 37°C. Thus the OE of the rat nasal cavity expresses HSP72, and this protein appears to play an important role in the ability of the tissue to withstand hyperthermia.

Keywords: Nasal cavity; Heat shock protein; Heat shock; Rat; Olfactory epithelium

Correlation between biomarkers of polycyclic aromatic hydrocarbon exposure and electrophilic tissue burden in a rat model by Adela Tzekova; Ross Thuot; Claude Viau (pp. 351-361).

This study was aimed at investigating the correlation between biomarkers of exposure to polycyclic aromatic hydrocarbons and, more specifically, at examining the role of urinary 1-hydroxypyrene (1-OHP) as a reliable measure of internal dose linked to the electrophilic tissue burden (ETB), assessed as covalent binding of the ultimate carcinogen benzo(a)pyrene diolepoxide (BaPDE) with cellular proteins in target organs. The protocol included experimental verification of a previously proposed algorithm for adjustment of reference values for urinary 1-OHP with exposure to different mixtures of polycyclic aromatic hydrocarbons in a rat model. Hence, the relationships between ETB in liver, lung, and heart as well as the BaPDE–haemoglobin adducts level on the one hand, and urinary/faecal 1-OHP or urinary/faecal 3-hydroxybenzo(a)pyrene (3-OHBaP) on the other hand have been examined. Male Sprague-Dawley rats received intraperitoneally, once daily for 10 consecutive days, binary mixtures of benzo(a)pyrene (BaP) and pyrene (P) in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5, with three doses of BaP (2, 6 and 20 mg/kg) for each scenario. The ETB levels were measured as the ultimate analyte benzo(a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE adducts with proteins. It was experimentally confirmed that: (1) urinary 1-OHP is a reliable biomarker linked to the ETB in tissues that are targets for carcinogenicity, such as lung, for the BaP/P ratios of 0.2 and 1 (linear regression p=0.0099 and 0.0293, respectively); (2) urinary 3-OHBaP is correlated with the BaPDE–haemoglobin adducts for all three exposure scenarios (p=0.0011 for BaP/P=0.2, p<0.0001 for BaP/P=1 and p=0.0099 for BaP/P=5). The experimental relationship between ETB and urinary 1-OHP was used to interpolate biological limit values for the urinary metabolite assuming three arbitrary critical levels of ETB. These were compared with the values calculated from the algorithm using the BaP/P ratio 1 mixture as a reference. The ratios of calculated to observed values varied from 1.0 to 1.6 for the BaP/P 0.2 mixture, and from 1.9 to 3.0 for the BaP/P 5 mixture. The results obtained in the present study indicate that the algorithm mentioned above applies well for two of the three exposure scenarios corresponding to realistic occupational BaP/P ratios of 0.2 and 1. This suggests that, using ETB as an endpoint, the proposed algorithm will reasonably predict the critical value of urinary 1-OHP for mixtures having different BaP/P ratios. Stronger linear relationships between ETB in all chosen tissues and 1-OHP or 3-OHBaP excretion were obtained with urinary metabolites than with their faecal analogues. Thus urinary 1-OHP and 3-OHBaP are more reliable biomarkers in biological monitoring strategies.

Keywords: Benzo(a)pyrene; 3-Hydroxybenzo(a)pyrene; Benzo(a)pyrene diolepoxide; Polycyclic aromatic hydrocarbon; Pyrene; 1-Hydroxypyrene; Electrophilic tissue burden

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Archives of Toxicology (v.78, #6)