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Archives of Toxicology (v.75, #9)

No Title by Ruth A. Roberts; Kenny S. Crump; Werner K. Lutz; Hans-Juergen Wiegand; Gary M. Williams; Paul T. Harrison; Iain F. Purchase (pp. 507-512).

The uncertainties that surround the methods used for risk assessment of exposure to carcinogens have been highlighted by a recent document advocating an approach based on the T25 dose (the dose giving a 25% incidence of cancer in an appropriately designed animal experiment). This method relies on derivation of the T25 dose then assesses risk at the exposure dose using proportionality provided by a linear extrapolation (T25/linear). To promote discussion of the scientific issues underlying methods for the risk assessment of chemical carcinogens, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) hosted a one-day workshop in Brussels on 10 November 2000. Several invited presentations were made to participants, including scientists from regulatory authorities, industry and academia. In general, it was felt that there was sufficient basis for using the T25 dose as an index of carcinogenic potency and hence as part of the hazard assessment process. However, the use of the T25 in risk assessment has not been validated. The T25/linear and other extrapolation methods based on metrics such as LED10 assume linearity which may be invalid. Any risk calculated using the T25/linear method would provide a precise risk figure similar to the output obtained from the Linearised Multi-stage (LMS) method formerly used by the Environmental Protection Agency (EPA) in the United States of America. Similarity of output does not provide validation but rather reflects their reliance on similar mathematical approaches. In addition to the T25 issue, evidence was provided that using two separate methods (linearised non-threshold model for genotoxic carcinogens; no-observable-effect level with a safety factor (NOEL/SF) method for all other toxicity including non-genotoxic carcinogens) is not justified. Since the ultimate purpose of risk assessment is to provide reliable information to risk managers and the public, there was strong support at the workshop for harmonisation of approaches to risk assessment for all genotoxic and nongenotoxic carcinogens. In summary, the T25 method has utility for ranking potency to focus efforts in risk reduction. However, uncertainties such as the false assumption of precision and non-linearity in the dose-response curve for tumour induction raise serious concerns that caution against the use of T25/linear method for predicting human cancer risk.

Keywords: T25 dose Carcinogen Risk assessment Threshold

No Title by Kazushi Okazaki; Shuzo Okazaki; Susumu Nishimura; Hideaki Nakamura; Yasuki Kitamura; Kazuhisa Hatayama; Atushi Nakamura; Toshiharu Tsuda; Tomoyoshi Katsumata; Akiyoshi Nishikawa; Masao Hirose (pp. 513-521).

In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of methoxychlor, a chlorinated hydrocarbon pesticide with pro-estrogenic and anti-androgenic activities. Attention was paid to the sensitivity of certain additional parameters for detecting endocrine related effects of endocrine disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were allocated to one of four groups, each consisting of ten males and ten females, and methoxychlor was administered once daily by gavage at doses of 0 (control), 20, 100 or 500 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during 4 days after the 28th administration. Male rats receiving methoxychlor showed mainly atrophy of mammary acinus in the 20 mg/kg and higher groups, together with decreases in prostate and seminal vesicle weights, and atrophy of epididymis, prostate, seminal vesicle and coagulating gland in the 100 and 500 mg/kg groups. In addition, decrease in serum testosterone level, increase in follicle-stimulating hormone level, decrease in testis and epididymis weights, atrophy of semiferous tubules and Leydig cells, decrease in the number of sperm in the caudal epididymis and their motility were observed in the 500 mg/kg group. Female rats receiving methoxychlor showed mainly abnormal estrous cycles, decrease in serum luteinizing hormone level, decrease in ovary weight, proliferation of mammary acinus, atrophy of ovary due to decrease in follicles and corpus luteum in histopathology, hypertrophy of endometrial epithelium of uterus and vagina epithelium in the 100 and 500 mg/kg groups. Among the parameters tested in the present experimental system, effects of methoxychlor on endocrine-related organs were detected with regard to serum hormone, organ weights, histopathological examination in both sexes, estrus cycle in females and sperm examination in males. Based on these results, a no-observed-adverse-effect level (NOAEL) in the present study was estimated to be below 20 mg/kg per day. In particular, the adverse effects were effectively detected in organ weights of accessory sex organs and histopathological examination.

Keywords: Methoxychlor Estrogen Rat Enhanced OECD test guideline 407 Endocrine disrupters

No Title by Gunilla Eklund; Kierstin Petersson Grawé; Agneta Oskarsson (pp. 522-530).

Infants are exposed to higher levels of cadmium (Cd) from infant and follow-on formulas than from breast milk. We studied the bioavailability of ¹⁰⁹CdCl₂ from cows' milk formula, soy formula, wheat/oat/milk formula, wholemeal/milk formula and water in 11-day-old rat pups. The pups received a single oral dose of one diet labelled with ¹⁰⁹Cd, 0.1 or 0.3 mg Cd/kg body weight. After 2 or 24 h or 4, 9 or 12 days the fractional retention of ¹⁰⁹Cd in the whole body, in segments of rinsed small intestine and in tissue was measured in a gamma counter. Pups receiving ¹⁰⁹Cd in water or cows' milk formula had the highest mean whole-body retention. It ranged from 67% of the dose in the water group to 52% in the wholemeal/milk formula group 4 days after dosing. The retention of ¹⁰⁹Cd in the rinsed small intestine was significantly higher in the water group and the cows' milk formula group than in the cereal-based formula groups at 24 h and 4 days after dosing. It was still high in all groups on day 9, ranging from 26 to 11%. Initially most of the ¹⁰⁹Cd was retained in the duodenum but by day 4 it had moved further down into the jejunum. In the liver, the highest and lowest retention on day 4 was 16‰ and 3‰ of the dose in the water group and wholemeal/milk formula group, respectively. In the kidney, ¹⁰⁹Cd was still increasing 12 days after exposure in all groups. Whole-body retention and tissue levels were higher than previously reported in adult animals. The lower bioavailability of ¹⁰⁹Cd from the cereal-based formulas compared to water and cows' milk formula on the longer survival times is most likely explained by Cd binding to dietary fibre and phytic acid in the cereal-based formulas reducing the intestinal binding and decreasing the bioavailability of Cd. The high retention of ¹⁰⁹Cd in the small intestine, leading to a prolonged absorption period, emphasizes the importance of extending studies on neonatal Cd absorption over a long time period in order to detect for example, endpoints, accumulation of Cd in the kidney.

Keywords: Absorption Cadmium Infant formula Newborn

No Title by Smarajit Maiti; Ajay K. Chatterjee (pp. 531-537).

Arsenic is a potent toxin, carcinogen and modulator of antioxidant defense system. In this study, male rats of Wistar strain, maintained on either 18% or 6% protein (casein) diet, received an acute i.p. exposure to sodium arsenite (As³⁺) at its LD₅₀ dose (15.86 mg/kg body weight). One hour after the arsenic exposure, glutathione (GSH) concentration was significantly depleted and lipid peroxidation was increased. A relationship between any two of tissue arsenic concentrations, GSH levels and lipid peroxidation values was observed only for liver when the proportional changes of respective parameters in either of the dietary groups of animals were compared. This suggests that, in liver, arsenic metabolism appears dependant upon the GSH concentration. Acute arsenic exposure significantly increased the glutathione peroxidase (GPx) activity in liver of both dietary groups and in kidney of only the 18% protein-fed group of animals. The glutathione-S-transferase (GST) activity significantly decreased in liver of the 18% protein-fed animals while GST increased in kidney of both the 18% and the 6% protein-fed groups. No significant change in glutathione reductase (GR) or glucose-6-phosphate dehydrogenase (G6PDH) activity was observed. In the present investigation, liver as a whole seems to be more affected in terms of GSH level and GST activity. The mode of responses of GPx and GR activities as well as the unaltered G6PDH activity might result in arsenic-induced GSH depletion and increase in lipid peroxidation. The animals of the 6% protein-fed group, appeared to be affected less in terms of tissue arsenic concentration, lipid peroxidation, GSH level and GST activity.

Keywords: Arsenic-treatment Low protein diet Liver Kidney Glutathione Antioxidant system.

No Title by Yasuhiro Masubuchi; Tomohisa Nakano; Atsushi Ose; Toshiharu Horie (pp. 538-543).

Oxidative metabolism of carbamazepine results in covalent binding of its reactive metabolite to liver microsomal proteins, which has been proposed as an important event in pathogenesis of the hypersensitivity reactions to this drug. Although the proposed reactive metabolites are produced by cytochrome P450 enzymes (P450 or CYP), the impact of the formation of unstable metabolites on the enzyme itself has not been elucidated. The present study examines the alteration of P450 enzyme activities during the metabolism of carbamazepine. Liver microsomes from rats and humans were preincubated with carbamazepine in the presence of NADPH, and subsequently assayed for monooxygenase activities representing several P450s. No evidence was obtained for inactivation of CYP2C11, CYP3A, CYP1A1/2 or CYP2B1/2 in rat liver microsomes during the carbamazepine metabolism, whereas the CYP2D enzyme was inactivated in a manner related to the preincubation time. Interestingly, under the same protocol human liver microsomes did not exhibit inactivation of CYP2D6, as well as there being no CYP2C8, CYP2C9 or CYP3A4 inactivation, whereas CYP1A2 was inactivated. Reduced glutathione could not protect against the observed inactivation of the P450s. These results suggest that CYP2D enzyme(s) in rats and CYP1A2 in humans biotransform carbamazepine into reactive metabolites, resulting in inactivation of the enzyme themselves, and raise the possibility that the P450 isoforms participate in toxicity induced by the drug in both animal species.

Keywords: Carbamazepine Cytochrome P450 Liver microsomes Reactive metabolite Species difference

No Title by Guofang Lin; Qingwen Ma; Jigang Chen; Jianhua Shen; Cuiqing Xiang; Klaus Golka; Dongsheng Zhang (pp. 544-548).

The distribution of the polymorphic alleles of the genes coding for glutathione S-transferases (GSTs) M1 and T1 was compared with the results of cytological grading of exfoliated urothelial cells (Pap test) in a non-diseased high-risk group of workers formerly exposed to benzidine in the Shanghai dyestuff industry (n=317). All subjects were genotyped for GSTT1 and M1 gene polymorphism by allele-specific PCR. Individuals were stratified according to their job and duration of exposure. A subgroup of 78 individuals with cytological gradings of grade III or higher in the Pap test showed a significant under-representation of the combination of GSTT1 0/0 and M1 0/0 genotypes compared with 238 subjects with a cytological classification lower than grade III (OR 0.55, 95% CI 0.31–0.98, P=0.04). These results suggest that neither the GSTM1 0/0 or GSTT1 0/0 genotype alone nor their combination had a clear association with cytopathological changes in exfoliated urothelial cells from individuals previously exposed to benzidine in Shanghai. This contradicts the results of studies indicating that the GSTM1 0/0 genotype is associated with an increased risk for bladder cancer in the general population, mostly outside China.

Keywords: Benzidine Dye industry Cytological grading Glutathione S-transferase T1 Glutathione S-transferase M1 Chinese

No Title by Nobuhiro Konno; Masashi Tsunoda; Ken Nakano; Yang Liu (pp. 549-554).

The purpose of this study was to determine the effect of tributyltin chloride (TBTCl) on the NMDA receptor by in vitro and in vivo experiments. In the first in vitro experiment, the binding of [³H]MK-801 and of [³H]-CGP39653 were studied in membrane preparations from the cerebral cortex of intact mice to obtain control values. Saturation experiments for [³H]MK-801 and [³H]CGP39653 revealed single binding sites with K _d values of 10.27 and 37.8 nM, and receptor densities of 1.75 and 2.20 pmol/mg of protein, respectively. In the second in vitro experiment, displacement studies were carried out with TBTCl over a concentration range of 0.1 µM to 2 mM. TBTCl inhibited [³H]MK-801 binding but did not affect [³H]CGP39653 binding. In the in vivo experiments, the mice received 1–125 ppm TBTCl in the diet ad libitum for 30 days. Ligand binding to cortical membrane preparations from each mouse was measured by a one-concentration point (2 nM) binding assay. [³H]MK-801 binding was significantly lowered (P<0.05) in the 5 and 125 ppm TBTCl-exposed animals compared with the controls. [³H]CGP39653 binding was also significantly lowered (P<0.05) in the 1 and 125 ppm TBTCl-exposed animals compared with the controls. These results suggest that the NMDA receptors in the mouse brain are sensitive to relatively low level exposure to TBTCl.

Keywords: Tributyltin NMDA receptor [3H]MK-801 [3H]CGP39653 Neurotoxicity

No Title by Toshinari Suzuki; Yoshio Nakagawa; Kuniaki Tayama; Kumiko Yaguchi; Tetsuya Suga (pp. 555-561).

The subacute toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic microsomal cytochrome P450 (P450) were investigated in male and female F344 rats. Rats were given 0.25, 0.5, and 1.0% terbutol for 28 days. Liver weights of male and female rats increased at all dose levels. The compound did not affect activity or amount of serum biochemical markers related to hepatic damage. The concentrations of terbutol in rat serum were less than 0.1 µM, and its major metabolites in serum were 2,6-di-tert-butyl-4-carboxyphenyl N-methylcarbamate and 2,6-di-tert-butyl-4-carboxyphenol. In male rats, P450 and cytochrome b ₅ (b ₅) contents, and NADPH cytochrome c reductase (fp2) activity in liver microsomes were increased about 2-fold by 1% terbutol administration for 7 to 28 days. Among the P450-dependent monooxygenase activities in liver microsomes, 7-benzyloxyresorufin-O-debenzylase (BROD) activity was greatly increased by 100-fold, and 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), and aminopyrine-N-demethylase (APND) activities were elevated 2- to 3-fold. 7-Methoxyresorufin-O-deethylase (MROD), erythromycin-N-demethylase (EMND), estradiol 2-hydroxylase (ED2H), chlorzoxazone 6-hydroxylase (CZ6H), and lauric acid ω-hydroxylase (LAOH) activities were unchanged. For the activities of testosterone hydroxylation, testosterone 16β-hydroxylase (T16BH) activity was markedly increased by 30-fold, and testosterone 6β-hydroxylase (T6BH) and testosterone 7α-hydroxylase (T7AH) activities were slightly elevated. Testosterone 2α-hydroxylase (T2AH) activity was not affected. Terbutol 4-methylhydroxylase (T4MH) activity was increased 9-fold by 1% terbutol. In an immunoinhibition study, T4MH activity in liver microsomes from 1% terbutol-treated rats was decreased about 50% by polyclonal anti-rat CYP2B1, whereas polyclonal anti-rat CYP2A1 and CYP2C11 did not affected the activity. These results indicate that terbutol increased CYP2B subfamily in rat liver microsomes, and that the compound did not cause serious hepatic damage.

Keywords: 2,6-Di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) Cytochrome P450 Rat liver microsomes Toxicity

No Title by Pablo Steinberg; Ingrid Zschaler; Elke Thom; Manuela Kuna; Günter Wüst; Angelika Schäfer-Schwebel; Rolf Müller; Peter-Jürgen Kramer; Günter Weiße (pp. 562-568).

7-Acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthaline (AHTN) is one of the two most widely used fragrances of a group of substances known collectively as the polycyclic musks. In the last few years evidence has been accumulating that AHTN is hepatotoxic when administered at high doses. In the present study the subchronic hepatotoxicity of AHTN administered to rats at doses within the human exposure range was evaluated. For this purpose female and male juvenile Wistar rats were exposed to AHTN (300 µg/kg body weight per day, i.p.) alone or to a single dose of diethylnitrosamine (DEN) (100 mg/kg body weight, i.p.) followed by AHTN (1, 10, 100 or 300 µg/kg body weight per day, i.p.) for 90 days. Thereafter the liver architecture as well as the presence of placental glutathione S-transferase (GST-P)-positive hepatic lesions was assessed. In male animals receiving AHTN alone or in combination with DEN the number of GST-P-positive single hepatocytes was similar to that in untreated rats, while GST-P-positive mini-foci and foci were not observed. In the case of female rats the number of GST-P-positive single hepatocytes and mini-foci in AHTN-treated rats was similar to that in untreated animals, whereas in those animals receiving AHTN either alone or in combination with DEN, GST-P-positive foci could not be detected or were present in a number as similar to that in untreated rats. In conclusion, in the present study it has been shown that AHTN administered over a 90-day period in concentrations similar to those taken up daily by humans does not lead to hepatotoxicity.

Keywords: 7-Acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthaline Hepatotoxicity Liver tumor initiation Liver tumor promotion

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Archives of Toxicology (v.75, #9)