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Archives of Toxicology (v.74, #3)
Repeated dose (28 days) oral toxicity study of flutamide in rats, based on the draft protocol for the `Enhanced OECD Test Guideline 407' for screening for endocrine-disrupting chemicals by Kazuhiro Toyoda; Makoto Shibutani; Toru Tamura; Takatoshi Koujitani; Chikako Uneyama; Masao Hirose (pp. 127-132).
In association with the international validation project to establish a test protocol for the `Enhanced OECD Test Guideline 407', we performed a preliminary 28-day, repeated-dose toxicity study of flutamide, a non-steroidal androgen antagonist, and assessed the sensitivity of a list of parameters for detecting endocrine-related effects of endocrine-disrupting chemicals (EDCs). Seven-week-old CD(SD)IGS rats were divided into four groups, each consisting of 10 males and 10 females, and administered flutamide once daily by oral gavage at doses of 0 (control), 0.25, 1 and 4 mg/kg body weight/day. Male rats were killed 1 day after the 28th administration. Female rats were killed on the day they entered the diestrus stage in the estrous cycle following the last treatment. Male rats receiving flutamide at dose levels of 1 and 4 mg/kg showed lobular atrophy of the mammary gland and a decrease in epididymal weight. In addition, 4 mg/kg flutamide-treated males exhibited raised serum testosterone and estradiol levels and decreased weight of the accessory sex glands. In females, a slight prolongation of the estrous cycle was also observed in the 4 mg/kg flutamide-treated group. No dose-related changes could be detected by haematology, serum biochemistry and sperm analysis. Thus, among the parameters tested in the present experimental system, the weight of endocrine-linked organs and their histopathological assessment, serum hormone levels, and estrous cycle stage allowed the detection of endocrine-related effects of flutamide.
Keywords: Key words Flutamide; Androgen antagonist; Rat; Enhanced OECD Test Guideline 407; Endocrine disrupters
Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey by Thomas G. Schulz; Frederike A. Wiebel; Ricarda Thier; Diether Neubert; Donald S. Davis; Robert J. Edwards (pp. 133-138).
The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy- 3-(p-nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30–42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.
Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats by Dai Mizuno; Einosuke Tanaka; Kozo Tanno; Shogo Misawa (pp. 139-144).
In this study we investigated cytochrome P450 (CYP) 2E1 expression using a probe drug, chlorzoxazone (CZX), whose metabolism can be used to monitor toluene exposure in rats. The animals received an i.p. injection of toluene (0.25, 0.5 and 1 ml/kg) once a day for 3 days. The total CYP and CYP2E1 content and the aniline and CZX hydroxylase activity (V max and CLint) increased depending on the dose of toluene administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from toluene-treated rats was reduced by about 80%. The IC50 values in microsomes from toluene-treated rats were between 3 and 5 μM. The production of HCZX and the activity of aniline hydroxylase in toluene-treated rats were correlated with the amount of rat CYP2E1 protein (r=0.88 and r=0.88, respectively). The elimination of CZX by toluene-treated rats was increased and the HCXZ production in the toluene-treated group was greater than that in the olive oil control group. The correlations between intrinsic clearance (CLint: V max/K m) in vitro and total body clearance (CLtot) of CZX hydroxylation and the elimination half-life (t 1/2) of CZX in vivo in toluene-treated rats were high (r=0.784, P < 0.001; r=−0.678, P < 0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and 2 h after CZX administration in vivo there was also a high correlation with CLint (V max/K m) in vitro (r=−0.729, P < 0.001). In conclusion, these results demonstrate that CZX is a very good probe for monitoring induction in toluene-treated rats.
Keywords: Key words Rat; Toluene; Cytochrome P450; Hepatic enzyme activity; Induction; Chlorzoxazone; CYP2E1
Effect of transforming growth factor-β1 on cytochrome P450 expression: inhibition of CYP1 mRNA and protein expression in primary rat hepatocytes by Gesine F. Müller; Olaf Döhr; Claudia El-Bahay; Regine Kahl; Josef Abel (pp. 145-152).
Primary hepatocytes are a widely used cell model to analyse the expression and regulation of hepatic cytochrome P450 (CYP) isoenzymes. Transforming growth factor-β1 (TGF-β1) was previously shown to inhibit constitutive and induced CYP1 expression in human cell lines and primary hepatocytes but not in rat cells. In the present study we examined the effect of TGF-β1 on constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced expression of CYP1 isoenzymes in primary rat hepatocytes in order to address the species-specificity of CYP1 down-regulation by TGF-β1. The results show an inhibition of TCDD-induced CYP1-related 7-ethoxyresorufin-O-deethylase (EROD), 7-methoxyresorufin-O-demethylase (MROD) activities and mRNA expression (determined by reverse transcriptase polymerase chain reaction, RT-PCR) by 100 pM TGF-β1 in cells co-treated for 24 h with 1 nM TCDD. However, while TGF-β1 also down-regulated constitutive EROD and MROD activities as well as CYP1A2 protein expression, it did not change the constitutive mRNA expression of CYP1 isoenzymes. The down-regulation seemed to be specific for CYP1 isoenzymes since constitutive expression of other CYP isoenzymes was unaffected concerning protein levels, as determined by Western blot for CYP2B1/2 and CYP3A1, as well as mRNA levels, as determined by RT-PCR for CYP2B1/2, CYP2E1 and CYP3A1. Thus, TGF-β1 not only inhibits CYP1 expression in humans but also in rats, indicating that regulation of CYP1 expression in these two species is similar.
Keywords: Key words CYP1A1; CYP1B1; Transforming growth factor TGF-β1; Rat hepatocytes; Reverse transcriptase polymerase chain reaction RT-PCR
Neurotoxic effect of L-2-chloropropionic acid on primary cultures of rat cerebellar granule cells by Nicholas C. Sturgess; Anne Rustad; Frode Fonnum; Edward A. Lock (pp. 153-160).
L-2-Chloropropionic acid (L-CPA), when administered orally to rats, produces selective necrosis to the granule cell layer of the rat cerebellum which is delayed in onset, not appearing until 36 – 48 h after exposure. The present study was conducted to characterise the toxic effect of L-CPA in primary cell cultures of rat cerebellar granule cells in vitro. Exposure to L-CPA produced a time and concentration dependent loss in cerebellar granule cell viability. Mean 50% effective concentration (EC50) values for L-CPA toxicity were 18.3 ± 0.3, 7.4 ± 0.1, and 3.5 ± 0.1 mM for 24, 48 and 72 h exposure respectively. Exposure for 24 h followed by a return to L-CPA free medium for 24 h was more toxic than exposure for 24 h alone. Cells maintained in culture for a longer duration were more susceptible to L-CPA-induced toxicity. The toxic effects of L-CPA could be partially or fully prevented by concomitant exposure of the cells to putative neuroprotective compounds. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (3 μM), afforded partial protection against L-CPA induced toxicity, whilst other glutamate receptor antagonists including, D(-)-2-amino-5-phosphopentanoic acid (D-AP5; 300 μM), D(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (D-CPP; 300 μM), 5,7-dichlorokynurenic acid (10 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 μM) were ineffective. The antioxidant, vitamin E (10 μM), afforded significant but incomplete protection from L-CPA toxicity. However when both MK-801 (3 μM) and vitamin E (10 μM) were present during L-CPA exposure, a greater degree of protection was observed than with either compound alone, although the combination failed to provide complete protection. Cyclosporin A, an inhibitor of the mitochondrial transition pore, also provided partial protection. By contrast, the free radical trapping agent, N-tert-butyl-α-(2-sulfophenyl)-nitrone (S-PBN) provided concentration (1–10 mM) dependent protection against the L-CPA-induced toxicity, which was complete at 10 mM. Our findings suggest that free radical production may be involved in the mechanism of L-CPA-induced toxicity.
Keywords: Key words L-2-Chloropropionic acid; Cerebellar granule cell toxicity-Excitotoxicity; Free radical species
Neurotoxicity of glutamate in chick telencephalon neurons: reduction of toxicity by preincubation with carbachol, but not by the endogenous fatty acid amides anandamide and palmitoylethanolamide by Mikael Andersson; Stig O. P. Jacobsson; Kent-Olov Jonsson; Gunnar Tiger; Christopher J. Fowler (pp. 161-164).
Exposure of chick telencephalon neurons in serum-free primary culture to glutamate produced a concentration-dependent cell toxicity as seen by an increase in lactate dehydrogenase (LDH) release that was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine and was reduced by preincubation with the cholinergic agonist carbachol. Preincubation with a threshold concentration of NMDA did not prevent glutamate toxicity, suggesting that chick NMDA receptors do not desensitize in the manner reported for their rodent counterparts. Neither anandamide (arachidonyl ethanolamide, AEA) nor palmitoylethanolamide (PEA) was able to prevent the neurotoxicity produced by prolonged glutamate incubation, even under conditions in which the metabolism of the compounds by fatty acid amide hydrolase or AEA cellular uptake was blocked. It is concluded that treatments reported as granting neuroprotection towards glutamate toxicity in rodent primary neuronal cultures do not necessarily show the same properties in the chick.
Keywords: Key words Carbachol; N-methyl-d-aspartate; Glutamate; Anandamide; Palmitoylethanolamide; Excitotoxicity; Chick; Neuronal culture
Efficacy of biperiden and atropine as anticonvulsant treatment for organophosphorus nerve agent intoxication by T.-M. Shih; J. H. McDonough (pp. 165-172).
The ability of the nerve agents tabun, sarin, soman, GF, VR, and VX to produce brain seizures and the effectiveness of the anticholinergics biperiden HCl or atropine SO4 as an anticonvulsant treatment were studied in a guinea-pig model. All animals were implanted a week prior to the experiment with cortical electrodes for electroencephalogram (EEG) recordings. On the day of exposure, the animals were pretreated with pyridostigmine (0.026 mg/kg, i.m.) 30 min prior to challenge with a 2 × LD50 dose (s.c.) of a given agent. In separate experiments, animals were challenged with 5 × LD50 (sc) of soman. One minute after agent challenge, the animals were treated intramuscularly (i.m.) with 2 mg/kg atropine SO4 admixed with 25 mg/kg 2-PAM Cl and then observed for the onset of seizure activity. Five minutes after the start of nerve agent-induced EEG seizures, animals were treated i.m. with different doses of biperiden HCl or atropine SO4 and observed for seizure termination. The anticonvulsant ED50 of biperiden HCl and atropine SO4 for termination of seizures induced by each nerve agent was calculated and compared. With equally toxic doses (2 × LD50) of these agents, continuous EEG seizures (status epilepticus) developed in all animals challenged with soman, tabun, or VR, and in more than 90% of the animals challenged with GF or sarin. In contrast, only 50% of the animals developed seizures when challenged with VX. The times to onset of seizures for soman, tabun, GF, and sarin were very similar (5–8 min) while for VR, it was about 10 min. In the case of VX, not only was the time to seizure development longer (20.7 min), but the seizure activity in 19% of the animals terminated spontaneously within 5 min after onset and did not return. Under these conditions, the anticonvulsant ED50s of biperiden HCl for soman, GF, VR, tabun, sarin, and VX were 0.57, 0.51, 0.41, 0.2, 0.1, and 0.09 mg/kg, respectively, while those of atropine SO4 for soman, VR, tabun, GF, sarin, and VX were 12.2, 11.9, 10.4, 10.3, 5.1, and 4.1 mg/kg, respectively. In separate experiments, the anticonvulsant ED50 doses of biperiden for animals challenged with 2 or 5 × LD50 of soman were 0.48 (95% confidence limits 0.25–0.73) or 0.57 (95% CI 0.38–0.84) mg/kg, respectively, while the anticonvulsant ED50s for atropine (12.2 mg/kg, i.m.) were identical under these same two challenge conditions. The present study demonstrates that all nerve agents can produce status epilepticus and that the therapeutic effectiveness of atropine and biperiden roughly paralleled the seizurogenic potential of these agents.
Keywords: Key words Organophosphorus compounds; Cholinesterase inhibitors; Soman; Sarin; Tabun; GF; VX; Convulsions; Seizures; EEG activity; Anticonvulsants; Atropine; Biperiden; Anticholinergic compounds
All-trans-retinoic acid and all-trans-retinoyl-β-d-glucuronide alter the development of axolotl embryos (Ambystoma mexicanum) in vitro by Renate Krätke; Ralph Rühl; Frank Kirschbaum; Heinz Nau (pp. 173-180).
Retinoids are involved in several physiological processes and are used in the treatment of various skin disorders. Therapy with retinoids during pregnancy may induce severe embryotoxic effects like craniofacial and cardiovascular malformations in the developing embryo. We investigated the effects of all-trans-retinoic acid (ATRA) and all-trans-retinoyl-β-D-glucuronide (ATRAG) in an amphibian embryotoxicity assay with Ambystoma mexicanum (axolotl) as an alternative in vitro method. Embryos were exposed to various concentrations of ATRA or ATRAG for 48 h beginning with the blastula stage. Kinetic investigations in the embryonic tissue were performed during the exposure period. Both retinoids interfered with the development of the axolotl embryos. Dose-dependent effects observed included growth retardation, craniofacial and cardiovascular malformations, as well as neural tube defects. In the axolotl, ATRA induced slightly more pronounced embryotoxic effects than ATRAG. All-trans-retinal was shown to be a major endogenous retinoid in this species. Endogenous levels of all-trans-retinaldehyde were increased during exposure to both ATRA and ATRAG. The glucuronide, however, was only detected in small amounts after ATRAG exposure. The embryotoxic potential of ATRAG could be explained by deglucuronidation to ATRA.
Keywords: Key words All-trans-retinoic acid; All-trans-retinoyl-β-d-glucuronide; Embryotoxicity; Amphibian assay; Alternative in vitro test