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Archives of Toxicology (v.76, #11)

Immature uterotrophic assay of estrogenic compounds in rats given diets of different phytoestrogen content and the ovarian changes with ICI 182,780 or antide by Kanji Yamasaki; Masakuni Sawaki; Shuji Noda; Takeharu Wada; Takaharu Hara; Mineo Takatsuki (pp. 613-620).

To investigate the influence of phyotestrogens in the diet, an immature uterotrophic assay of ethinylestradiol, bisphenol A, 4-nonylphenol or genistein was performed in rats given the formula MF diet, modified NIH-07 open formula diet, or modified NIH-07 phytoestrogen-lowered-diet (study 1). The chemicals were administered subcutaneously from 20 days of age for 3 days. Doses of ethinylestradiol, bisphenol A, 4-nonylphenol or genistein were 0.06–0.6 µg/kg per day, 1–10 mg/kg per day, 10–100 mg/kg per day or 1–20 mg/kg per day, respectively. In another study, an immature uterotrophic assay of genistein and ethinylestradiol together with ICI 182,780 or antide was performed to compare the ovarian changes with these chemicals (study 2). Doses of genistein or ethinylestradiol were 30 mg/kg per day or 0.6 µg/kg per day, respectively, and these chemicals were injected subcutaneously from 20 days of age for 3 days. In study 1, there were no essential differences in the uterus weights among the various phytoestrogen-content diets. In study 2, the ovary weights in rats given genistein were significantly higher than in the controls, whereas the ovary weights in rats given ethinylestradiol were lower than in the controls. The ovary weights in the ICI 182,780 plus genistein group were significantly higher than in the genistein group, but decrease of the ovary weights was detected in the antide plus genistein group. There was no significant difference in ovary weights between the ICI 182,780 plus ethinylestradiol group and the ethinylestradiol group, but decrease of ovary weights was detected in antide plus ethinylestradiol group. In a histological examination of the ovary, fluid-filled follicles in the genistein group were more numerous than in other groups and increase of granulosa cell fragmentation was seen in the ethinylestradiol and other groups with the exception of the genistein group. The present findings demonstrate that the sensitivity of the immature rat uterotrophic assay is not influenced by the relatively low level of phytoestrogen in diets and that the ovarian changes occurring with genistein and ethinylestradiol are different.

Keywords: Rodent diet Phytoestrogen Genistein Ethinylestradiol Rat Uterotrophic assay

Increased expression and decreased activity of cytochrome P450 1A1 in a murine model of toluene diisocyanate-induced asthma by Muriel Haag; Thea Leusink-Muis; Renaud Le Bouquin; Frans P. Nijkamp; Alain Lugnier; Nelly Frossard; Gert Folkerts; Françoise Pons (pp. 621-627).

In the lung, cytochromes P450 (CYP) may be affected by inhaled pollutants. In a previous study, we showed that acute inhalation of toluene diisocyanate (TDI), a low molecular weight chemical known to cause occupational asthma, decreased CYP2B1 expression in rat lung. In the present work, we investigated the effect of TDI in a murine model of TDI-induced asthma. Mice were skin-sensitized with TDI on 2 consecutive days and challenged intranasally 8 days later. Lung expression and activity of CYP were assessed 24 h after the challenge. A significant increase in Cyp1a1 protein expression was detected by western blotting in lung from mice sensitized and challenged with TDI, whereas no modification of expression of other CYP, namely Cyp2b, Cyp2e1 and Cyp3a was observed. Increase in Cyp1a1 protein was associated with an increase in Cyp1a1 mRNA, as assessed by polymerase chain reaction after reverse transcription of total lung RNA. However, a decreased Cyp1a1 activity, as measured by O-deethylation of ethoxyresorufin was observed in lung from TDI-sensitized and challenged mice, suggesting that TDI may inhibit Cyp1a1 function. In agreement with this hypothesis, in vitro experiments conducted on liver microsomes overexpressing Cyp1a1 after treatment of mice with 3-methylcholanthrene showed that TDI markedly inhibited in a concentration-dependent manner Cyp1a1 activity. In conclusion, expression of Cyp1a1, known to exhibit rather negative functions in the lung, is increased in mice sensitized and challenged with TDI. However, this effect is associated with a decreased enzyme activity, which might limit the toxicological consequences of increased Cyp1a1 expression.

Keywords: Toluene diisocyanate Cytochrome P450 CYP1A1 Lung Asthma

Fractionation of protein adducts in rats and mice dosed with [¹⁴C]pentachlorophenol by Chin-Hsiang Tsai; Po-Hsiung Lin; Suramya Waidyanatha; Stephen M. Rappaport (pp. 628-633).

Pentachlorophenol (PCP) induces liver cancer in mice, possibly due to covalent binding of PCP metabolites to critical macromolecules. In this work, covalent binding was related to PCP biotransformation and specific (cysteinyl) adducts of chlorinated quinones in liver and blood of Sprague-Dawley rats and B6C3F1 mice dosed with [¹⁴C]PCP. Using a sequential scheme of scintillation counting along with selective cleavage of cysteinyl adducts by Raney nickel, we quantified total radiobinding, total covalent binding, non-cysteinyl protein binding, and specific protein adducts in liver nuclei (Np), liver cytosol (Cp), hemoglobin (Hb), and serum albumin (Alb). Almost all of the radiobinding to Np (>98%) was attributed to covalent binding in both rats and mice. Regarding Cp, more covalent binding was observed in mice than in rats (100% versus 67%, P=0.015). Very little binding was attributed to serum Alb (rats 1.3%, mice 2.6%, P=0.046) or Hb (not detected in either species). These results indicate that the liver was the main organ for PCP metabolism and that relatively little of the dose of reactive metabolites became systemically available. Cysteinyl binding accounted for 76–91% of total covalent binding to Np and 68–76% of total covalent binding to Cp. In addition, five times more PCP was bioactivated in the livers of mice than in those of rats (2.14% of the dose bound to Cp in mice and 0.416% in rats). These results reinforce previous studies, suggesting that the liver was a target organ of PCP carcinogenicity and that mice were more susceptible to liver damage than rats. However, the sum of all quantified adducts accounted for only 7–8% of total cysteinyl binding to Np and 2% to Cp, suggesting that other uncharacterized binding species may be important to the toxicity of PCP.

Keywords: Pentachlorophenol Raney nickel Protein binding Tetrachlorobenzoquinone

Phenotyping of human glutathione S-transferase hGSTT1-1: a comparison of two ex vivo routine procedures by Michael Müller; Jürgen Bünger; Michael Voss; Götz Westphal; Peter Ruhnau; Ernst Hallier (pp. 634-642).

The human glutathione S-transferase hGSTT1-1 is characterized by a polymorphism displaying three phenotypes: the "non-conjugator" (NC) phenotype expresses no or only a residual activity due to a homozygous deletion of the hGSTT1 gene, a medium hGSTT1-1 activity can be demonstrated for the "low conjugator" (LC) phenotype as the heterozygous bearer of one hGSTT1 allele, and a high hGSTT1-1 activity is detected for the "high conjugator" (HC) phenotype as the homozygous bearer of two hGSTT1 alleles. We have developed a routine ex vivo photometric phenotyping procedure based on the determination of bromide release rates from the hGSTT1-1-catalyzed glutathione conjugation of the substrate methyl bromide in EDTA blood samples under standard conditions (1,000 ppm methyl bromide, 10 min incubation). The bromide release rates were standardized to the hemoglobin (Hb) value. Twenty-six individuals were phenotyped following the new procedure. Four individuals were classified as NCs (24–33 pmol Br^–/mg Hb per min), 21 individuals were regarded as LCs (107–206 pmol Br^–/mg Hb per min) and one person of the study group was designated HC (236 pmol Br^–/mg Hb per min). The results were validated by qualitative hGSTT1 genotyping and demonstrated a 100% match for conjugators and non-conjugators. A second HPLC phenotyping routine procedure based on the formation of S-methylglutathione from methyl chloride in erythrocyte lysate incubations (Müller et al. 2001, Arch Toxicol 74:760–767) was established and validated by genotyping. The phenotyping results obtained with both methods were correlated, resulting in a good correlation with R ²=0.64 (y=0.8997x+51.535). Three distinct phenotype clusters for NCs, LCs and HCs, consistent with the proposed genetics, were demonstrated. Assay-dependent storage experiments revealed an excellent stability of the hGSTT1-1 activity. In conclusion, the evaluated methods provide powerful tools for determination of hGSTT1-1 activity as a clinical parameter.

Keywords: Glutathione S-transferase T1 Phenotyping Photometric assay HPLC method Halomethanes

Cytochrome P450 CYP1B1 and catechol O-methyltransferase (COMT) genetic polymorphisms and breast cancer susceptibility in a Turkish population by Neslihan Kocabaş; Semra Şardaş; Suzanne Cholerton; Ann K. Daly; Ali Karakaya (pp. 643-649).

Epidemiological studies indicate that most risk factors for breast cancer are related to reproductive and hormonal factors. Estrogen has been proposed to trigger breast cancer development via an initiating mechanism involving its metabolite, catechol estrogen (CE). Because of the important role of cytochrome P450 1B1 (CYP1B1) and catechol O-methyltransferase (COMT) in mammary estrogen and carcinogen metabolism, we examined the CYP1B1 and COMT genes to determine whether genetic variations could account for inter-individual differences in breast cancer. In this case-control study, we determined CYP1B1 and COMT genotypes in 84 breast cancer patients and 103 healthy unrelated women controls from a Turkish population. In the case of CYP1B1, we genotyped CYP1B1*3 (L432 V) allele. We found that carriers of the CYP1B1*3 allele were more frequent among breast cancer patients with adjusted odds ratio (OR) for age, age at menarche, age at first full-term pregnancy, body mass index (BMI) and smoking status of 2.32 (95% confidence interval 1.26–4.25) associated with the allele. However, this allele appeared to be a significant factor for susceptibility only in patients with a BMI greater than 24 kg/m². Menopausal status did not appear to affect susceptibility. In the case of COMT, there was no significant difference in susceptibility for breast cancer development between patients with low activity COMT-L (V158 M) allele and high activity COMT-H allele, and susceptibility was not affected by menopausal status, BMI or CYP1B1 genotype. We conclude that the CYP1B1*3 allele appears to be a factor for susceptibility to breast cancer in Turkish women especially those with a BMI greater than 24 kg/m².

Keywords: Breast cancer CYP1B1 COMT Genetic polymorphisms Estrogen Turkish population

Therapeutic efficacy of the adenosine A₁ receptor agonist N ⁶-cyclopentyladenosine (CPA) against organophosphate intoxication by Tjerk J. Bueters; Bas Groen; Meindert Danhof; Ad P. IJzerman; Herman P. van Helden (pp. 650-656).

The objective of the present study was to investigate whether reduction of central acetylcholine (ACh) accumulation by adenosine receptor agonists could serve as a generic treatment against organophosphate (OP) poisoning. The OPs studied were tabun (O-ethyl-N-dimethylphosphoramidocyanidate), sarin (isopropylmethylphosphonofluoridate), VX (O-ethyl-S-2-diisopropylaminoethylmethylphosphonothiolate) and parathion (O,O-diethyl-O-(4-nitrophenyl)phosphorothioate). The efficacy of the adenosine A₁ receptor agonist N ⁶-cyclopentyladenosine (CPA) against an OP intoxication was examined on the basis of the occurrence of clinical symptoms that are directly associated with such intoxication. CPA (1–2 mg/kg) effectively attenuated the cholinergic symptoms and prevented mortality in lethally tabun- or sarin-intoxicated rats. In contrast, CPA (2 mg/kg) proved to be ineffective against VX or parathion intoxication. Intracerebral microdialysis studies revealed that survival of sarin-poisoned and CPA-treated animals coincided with a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA. In VX-intoxicated rats, however, the ACh amounts increased 18-fold, irrespective of treatment with CPA. The striatal acetylcholinesterase (AChE) activity following a lethal sarin intoxication was completely abolished in the vehicle-treated animals, whereas 10% and 60% AChE activity remained in animals treated with 2 mg/kg CPA 1 min after or 2 min prior to the poisoning, respectively. In VX-intoxicated animals the AChE activity in the brain was strongly reduced (striatum 10%, hippocampus 1%) regardless of the CPA treatment. These results demonstrate that CPA is highly effective against tabun or sarin poisoning, but fails to protect against VX or parathion. Survival and attenuation of clinical signs in tabun- or sarin-poisoned animals are associated with a reduction of ACh accumulation and with protection of AChE activity in the brain.

Keywords: Organophosphate N6-Cyclopentyladenosine Acetylcholine release Adenosine A1 receptor

Effects of 5-day styrene inhalation on serum prolactin and dopamine levels and on hypothalamic and striatal catecholamine concentrations in male rats by Hubertus Jarry; Maria Metten; Armin O. Gamer; Wolfgang Wuttke (pp. 657-663).

In several studies a hypersecretion of the pituitary hormone prolactin (PRL) in styrene-exposed workers has been described. This should cause reproductive problems like oligomenorrhea, secondary amenorrhea and reduced fertility [Arfini et al. (1987) J Occup Med 29:826–830, Bergamaschi et al. (1996) Neurotoxicology 17:753–760, Mutti and Smargiassi (1998) Toxicol Ind Health 14:311–323]. Secretion of PRL is tonically inhibited by the catecholamine dopamine (DA), which is released from hypothalamic neurons. It has been suggested that the activity of the enzyme dopamine-β-hydroxylase (DBH) in the serum is a peripheral marker of central dopaminergic function. A slight reduction of such enzymatic activity was observed in styrene-exposed workers, which was associated with hypersecretion of PRL. To further investigate the putative effects of styrene on PRL release, male rats were exposed to styrene vapors (645, 2150 and 6450 mg/m³) for 6 h/day on 5 consecutive days. Animals were killed either directly following the last exposure (immediate group) or after a recovery period of 24 h (recovery group). Serum PRL and DA levels were measured by radioimmunoassay. Concentrations of catecholamines and their metabolites in the striatum and mediobasal hypothalamus (MBH) were determined by high performance liquid chromatography with electrochemical detection. Neither in the immediate nor in the recovery group were any statistically significant changes of serum PRL levels observed. Likewise, concentrations of catecholamines and their metabolites in the striatum and MBH remained unaffected. We conclude from these data that styrene, even at very high concentrations, has no adverse effects on the neuroendocrine mechanisms regulating PRL release and DA levels in the brain. With the limitations inherent in any animal model, we suggest that our data indicate that styrene also has no adverse neuroendocrine effects in humans.

Keywords: Styrene Inhalation exposure Prolactin Catecholamines Hypothalamus

Inhibitory effect of berberine on tert-butyl hydroperoxide-induced oxidative damage in rat liver by Jin-Ming Hwang; Chau-Jong Wang; Feu-Pi Chou; Tsui-Hwa Tseng; Yih-Shou Hsieh; Wea-Lung Lin; Chia-Yih Chu (pp. 664-670).

Berberine, a main protoberberine component of Coptidis Rhizoma, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in rat liver. In the preliminary study, berberine expressed an antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Further investigations were conducted using t-BHP-induced cytotoxicity in rat primary hepatocytes and hepatotoxicity in rats to evaluate the antioxidative bioactivity of berberine. The results in rat primary hepatocytes demonstrated that berberine, at the concentrations of 0.01–1.0 mM, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM). Berberine also attenuated the t-BHP-induced depletion of glutathione (GSH) and induced a high level of DNA repair synthesis. The in vivo study showed that the intraperitoneal pretreatment with berberine (0.5 and 5 mg/kg) for 5 days before a single dose of t-BHP (0.1 mmol/kg) significantly lowered the serum levels of hepatic enzyme markers (ALT and aspartate aminotransferase) and reduced oxidative stress in the liver. The histopathological evaluation of the livers revealed that berberine reduced the incidence of liver lesions, including hepatocyte swelling, leukocyte infiltrations, and necrosis induced by t-BHP. These results lead us to speculate that berberine may play a chemopreventive role via reducing oxidative stress in living systems.

Keywords: Berberine tert-Butyl hydroperoxide Cytotoxicity Genotoxicity Hepatocyte

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Archives of Toxicology (v.76, #11)