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Archives of Toxicology (v.77, #9)

Tamoxifen: 28-day oral toxicity study in the rat based on the Enhanced OECD Test Guideline 407 to detect endocrine effects by Philippe Kennel; Catherine Pallen; Erio Barale-Thomas; Gemma Espuña; Remi Bars (pp. 487-499).

The main objective of this 28-day oral gavage toxicity study in the rat was to investigate which of the current and/or additional parameters of the OECD Test Guideline 407 would reliably and sensitively detect the endocrine-mediated effects of the nonsteroidal antiestrogen tamoxifen. In addition, as this study was performed using two subgroups of five animals of each sex run concurrently, it enabled an assessment of the intralaboratory reproducibility while also assessing the potential value of using ten animals of each sex per group instead of using the standard five animals of each sex per group stipulated by the current guideline. Tamoxifen was administered daily by gavage to groups of 7-week-old Wistar rats for at least 28 days at dose levels of 5, 30, or 200 µg/kg body weight. Additional parameters specified in the enhanced OECD Test Guideline 407 were spermatozoa enumeration and morphology of the cauda epididymis, hormonal analysis of the thyroid-stimulating hormone (TSH), triiodothyronine (T₃) and thyroxine (T₄) levels, monitoring of the estrous cycle during week 4 of treatment to ensure females were in diestrus on the day of terminal sacrifice, organ weight of ovary, uterus, thyroid gland, prostate gland (ventral and dorsolateral parts), seminal vesicles with coagulation glands and pituitary gland, and microscopic investigation of the pituitary gland, vagina, mammary gland, seminal vesicles with coagulating glands, epididymis, and prostate gland (ventral and dorsolateral parts). Overall, 200 µg/kg per day was considered to be the Maximum Tolerated Dose (MTD) in both sexes, resulting in a marked reduction of body weight gain, together with slight effects on clinical signs, hematology, plasma chemistry, and microscopic changes in some endocrine tissues. Five micrograms per kilogram per day represented the No Observed Adverse Effect Level (NOAEL) in males and the No Observed Effect Level (NOEL) in females. At the intermediate dose level (30 µg/kg per day), the current OECD Test Guideline 407 was appropriate to detect the specific endocrine-related changes induced by tamoxifen in females, based on the histopathology findings observed in the ovary and the uterus. The additional parameters which were found to be changed in females (thyroid hormone levels, ovary and uterus weights, and histopathology of vagina) provided supplementary information further confirming tamoxifen-mediated endocrine effects. In males, when data from the current Test Guideline 407 were considered at the intermediate dose level, specific endocrine effects were only indicated on the basis of the histopathology findings observed in the prostate gland. The additional parameters examined which were found to be changed (prostate gland and seminal vesicle weights, and histopathology of seminal vesicle and mammary gland) were necessary to confirm the specific tamoxifen-mediated endocrine effects. Hence, amongst the additional parameters contained in the enhanced OECD Test Guideline 407, organ weights and histopathological examination of endocrine-related organs were the most helpful in confirming the detection of tamoxifen-mediated endocrine effects. The reproducibility evaluation showed that a group size of five animals of each sex consistently allowed the detection of endocrine effects with the current Test Guideline in both sexes at the high dose level and in females at the intermediate dose level. Doubling the animal number from five to ten of each sex per dose level did not notably increase the sensitivity of detection of endocrine-mediated effects.

Keywords: Enhanced OECD Test Guideline 407; Subacute rodent toxicity study; Detection of endocrine effects; Tamoxifen; Antiestrogen

Hepatoprotective activity of polyhydroxylated 2-styrylchromones against tert-butylhydroperoxide induced toxicity in freshly isolated rat hepatocytes by Eduarda Fernandes; Márcia Carvalho; Félix Carvalho; Artur M. S. Silva; Clementina M. M. Santos; Diana C. G. A. Pinto; José A. S. Cavaleiro; Maria de Lourdes Bastos (pp. 500-505).

2-Styrylchromones are a novel class of chromones, vinylogues of flavones (2-phenylchromones), which have recently been found in nature. The best described property of almost every group of flavones and other flavonoids, especially the hydroxylated derivatives, is their capacity to act as antioxidants. Indeed there is a widely accepted view that the positive health effects of flavones are due to their antioxidant activity. As oxidative stress is a main cause of liver toxicity induced by several hepatotoxicants, agents with the ability to protect the liver against reactive pro-oxidant species may be therapeutically useful. The present study evaluated the possible protective activity of six new synthetic polyhydroxylated 2-styrylchromone derivatives against the pro-oxidant hepatotoxicity exerted by tert-butylhydroperoxide (t-BHP) in freshly isolated rat hepatocytes. The cells were preincubated with the 2-styrylchromones in the final concentrations of 3.125, 12.5, 25, 50, 100, and 200 µM for 5 min before treatment with 1.0 mM t-BHP for 30 min (throughout this incubation period the cells were exposed to both compounds). The well-known antioxidant 3-hydroxyflavone (quercetin) was used as positive control. At the end of the 30-min incubation period, aliquots of cells suspensions were taken for measurement of lactate dehydrogenase leakage, thiobarbituric acid reactive substances, reduced glutathione, and oxidized glutathione contents. The tested compounds exhibited in vitro protective activity against t-BHP induced hepatotoxicity (1.0 mM, 30 min). Three of the tested compounds, at the concentrations of 3.125, 12.5, 25, and 50 µM, prevented the t-BHP induced glutathione depletion, lipid peroxidation, and cell death in freshly isolated rat hepatocytes to a comparable potency with that of quercetin.

Keywords: 2-Styrylchromones; tert-Butylhydroperoxide; Hepatoprotective activity; Freshly isolated rat hepatocytes

The effect of quercetin on the mRNA expression of different antioxidant enzymes in hepatoma cells by Elke Röhrdanz; Achim Bittner; Quynh-Hoa Tran-Thi; Regine Kahl (pp. 506-510).

The flavonol quercetin shows a wide range of effects in biological systems. We investigated whether quercetin exerts its proposed antioxidant properties via the antioxidant enzyme system. Quercetin in a concentration range from 5 to 100 µM decreased manganese superoxide dismutase, glutathione peroxidase, and copper zinc superoxide dismutase mRNA expression levels each by 30–40% in rat hepatoma H4IIE cells. Catalase mRNA expression levels increased about 30% but only with the cytotoxic concentration of 100 µM. Despite the down-regulation of antioxidant enzyme mRNA expression quercetin treatment of cells induced only a mild oxidative stress. Pretreatment of H4IIE cells with quercetin even protected against an oxidative stress resulting from hydrogen peroxide exposure. In conclusion, the antioxidant capacity of quercetin was shown not to be due to the antioxidant enzyme system.

Keywords: Quercetin; Catalase; Superoxide dismutase; Glutathione peroxidase; Hepatoma cells

Forestomach tumor induction by 2,4-hexadienal in F344N rats and B6C3F1 mice by Po C. Chan; Joel Mahler; Shyamal Peddada; Liat Lomnitski; Abraham Nyska (pp. 511-520).

2,4-Hexadienal (2,4-Hx) was studied for its toxicity and carcinogenicity because of its α, β-unsaturated aldehyde structure and potential link between exposure to lipid peroxidation products in the diet and human malignancies. Male and female F344N rats and B6C3F1 mice received 2,4-Hx in corn oil by gavage for 16 days, 14 weeks, or 2 years. In the 16-day studies 2,4-Hx induced forestomach necrosis and ulceration at 240 mg/kg and forestomach epithelial hyperplasia at 80 mg/kg in rats and mice. In the 14-week studies the chemical induced forestomach hyperplasia and nasal olfactory atrophy or necrosis at 120 mg/kg in rats and mice. In the 2-year studies 2,4-Hx induced squamous cell papilloma and carcinoma of the forestomach in male and female rats at 45 and 90 mg/kg and in male and female mice at 120 mg/kg. Two male mice in the 120 mg/kg group had uncommon squamous cell carcinoma of the oral cavity (tongue). Mechanistic studies indicated that the forestomach carcinogenesis in rats and mice may be due to depletion of glutathione as a result of oxidative stress induced by 2,4-Hx.

Keywords: 2,4-Hexadienal; Forestomach tumors; Rats; Mice

Ultrastructural changes induced by the des-F(6)-quinolone garenoxacin (BMS-284756) and two fluoroquinolones in Achilles tendon from immature rats by Mehdi Shakibaei; Ralf Stahlmann (pp. 521-526).

Garenoxacin is a des-(6)-fluoroquinolone exhibiting a comparatively low chondrotoxic potential in juvenile animals. We studied the effects of the drug on Achilles tendons in immature Wistar rats treated by oral intubation once daily (1) for 5 consecutive days from postnatal week 4 onward at doses of 0 (vehicle), 200 and 600 mg/kg body weight (b wt), and (2) for 21 consecutive days from postnatal day 4 onward at doses of 0 (vehicle), 80, 240 or 300 mg/kg b wt; ofloxacin or ciprofloxacin were used as comparators. Achilles tendon specimens were studied by electron microscopy. In comparison with vehicle-treated controls, ultrastructural changes were detectable in all samples from the garenoxacin-, ofloxacin-, or ciprofloxacin-treated rats (one animal per group). We found degenerative changes such as multiple vacuoles and large vesicles in the cytoplasm of tenocytes that resulted from swelling and dilatation of cell organelles (mitochondria, endoplasmic reticulum), densified nuclei and clumped chromatin; furthermore, cells that detached from the extracellular matrix, a general decrease of the fibril diameter and an increase in the distance between the collagenous fibrils were recognizable. The degree of changes increased with increasing doses. It remains unclear what these findings mean with respect to a possible risk in juvenile patients treated with garenoxacin or the other quinolones, but our results underline the fact that, in principle, this des-(6)-fluoroquinolone also has the potential to cause changes in connective tissue structures.

Keywords: Fluoroquinolones; Achilles tendon; Electron microscopy; Rat

Nephrotoxic effects of lead nitrate in Rana ridibunda by N. S. Loumbourdis (pp. 527-532).

The impact of lead (Pb) on kidney histopathology of the frog Rana ridibunda was investigated. Female frogs were exposed for 4, 10 and 30 days to 14 ppm lead (as lead nitrate). All the lead concentrations and many histological changes were time dependent. Light microscopy of kidney revealed morphological changes mainly in the proximal convoluted tubule (PCT) cells. The most severe changes such as vacuolation, Perl's stained material, infiltration, brush border destruction and proximal tubule damage were detected in the animals exposed for 10 and 30 days. Karyomegaly was highest at 10-days exposure, probably as a result of intense stress caused by the lead. Some PCT in the 30-days-exposed animals were von Kossa's method positive, suggesting the presence of calcium. The possibility is discussed that some of these changes, such as karyomegaly and intranuclear inclusions, might be preneoplastic if lead was supplied at high concentrations and for long time.

Keywords: Frog; Lead; Histopathology; Kidney

Supplemental role of the Ames mutation assay and gap junction intercellular communication in studies of possible carcinogenic compounds from diesel exhaust particles by Edgar Rivedal; Oddvar Myhre; Tore Sanner; Ingvar Eide (pp. 533-542).

This study presents a new strategy for the carcinogenic evaluation of complex chemical mixtures based on genotoxic and nongenotoxic assays. We studied the ability of organic extracts of diesel exhaust particles (DEP) to induce point mutations in five different Salmonella typhimurium strains (Ames test) and to inhibit gap junction intercellular communication (GJIC) in rat liver epithelial cell lines. A crude extract of DEP was prepared by extraction with dichloromethane (DCM), and fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), nitro-PAH, dinitro-PAH, and polar compounds. Statistical experimental design, multivariate data analysis, and modeling were used to quantify the mutagenicity of individual and combined DEP fractions in the Ames assay. Quantitative determination of GJIC was carried out using a recently described combination of scrape loading and digital image analysis. Both assays responded to the DEP extract, but the responses were due to different fractions. The nitro-PAH fraction showed the strongest mutagenic potential, followed by the dinitro-PAH fraction. The effect on GJIC was due to the fraction containing the polar components, followed by the dinitro-PAH fraction. The extract was found to induce both basepair substitutions and frameshift mutations, through activation by bacterial nitroreductases. Hyperphosphorylation of connexin43, the major connexin in the epithelial cell lines, was less evident for DEP extract than for other communication inhibitors such as phorbol esters and growth factors, and consequently inhibitors of the protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathway were unable to counteract the inhibition by DEP extract. Since the Ames test is a well accepted method to screen for substances with genotoxic activity while inhibition of GJIC is associated with effect of tumor promoters and nongenotoxic carcinogens, it is not surprising but encouraging and interesting that the present data indicate that the two endpoints supplement each other as screening tests and in the evaluation of hazardous compounds in complex mixtures.

Keywords: Complex mixtures; Diesel exhaust particles; Gap junction intercellular communication; Multivariate data analysis; Nongenotoxic carcinogens

Modification of human serum albumin by acrylamide at cysteine-34: a basis for a rapid biomonitoring procedure by D. Noort; A. Fidder; A. G. Hulst (pp. 543-545).

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Archives of Toxicology (v.77, #9)

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Archives of Toxicology (v.77, #9)