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Archives of Toxicology (v.80, #12)

Repeated 28-day oral toxicity study of ketoconazole in rats based on the draft protocol for the “Enhanced OECD Test Guideline No. 407” to detect endocrine effects by Jae-Ho Shin; Hyun Ju Moon; Il Hyun Kang; Tae Sung Kim; In Young Kim; In Sook Park; Hyung Sik Kim; Eui Bae Jeung; Soon-Young Han (pp. 797-803).

We performed a 28-day repeated-dose toxicity study of ketoconazole, a widely used an antimycotic drug, based on the draft protocol of the “Enhanced OECD Test Guideline 407” (Enhanced TG407) to investigate whether ketoconazole has endocrine-mediated properties according to this assay. Seven-week-old SD rats were administered with ketoconazole daily by oral gavage at doses of 0, 6.25, 25 or 100 mg kg⁻¹ day⁻¹ for at least 28 days. The ketoconazole-treated male rats showed reduction of epididymis and accessory sex organ weights, spermatid retention in the seminiferous tubules, decrease of testosterone and increases of estradiol, luteinizing hormone (LH) and follicular stimulating hormone (FSH). A prolongation of the estrous cycle and increases of estradiol, LH and FSH were observed in the treated female rats. Thyroxin and triiodothyronine were decreased and thyroid-stimulating hormone was increased in both sexes; however, there were no compound-related microscopic lesions in the thyroid gland or changes in the thyroid weight. The endocrine-related effects of ketoconazole could be detected by the parameters examined in the present study based on the Organization for Economic Cooperation and Development (OECD) protocol, suggesting that the Enhanced TG407 protocol should be a suitable screening test for detection of endocrine-mediated effects of chemicals.

Keywords: Enhanced OECD Test Guideline 407; Ketoconazole; Rat; Endocrine disruptors

ATM/ATR-related checkpoint signals mediate arsenite-induced G₂/M arrest in primary aortic endothelial cells by Tsui-Chun Tsou; Feng-Yuan Tsai; Szu-Ching Yeh; Louis W. Chang (pp. 804-810).

Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular disease. Our recent in vitro studies have linked this vascular damage to vascular endothelial dysfunction induced by arsenic exposure. However, cell-cycle arrest induced by arsenic and its involvement in vascular dysfunction remain to be clarified. In this study, we employed primary porcine aortic endothelial cells to investigate regulatory mechanisms of G₂/M phase arrest induced by arsenite. Our study revealed that lower concentrations of arsenite (1 and 3 μM) increased cell proliferation, whereas higher concentrations of arsenite (10, 20, and 30 μM) inhibited cell proliferation together with correlated increases in G₂/M phase arrest. We found that this arsenite-induced G₂/M phase arrest was accompanied by accumulation and/or phosphorylation of checkpoint-related molecules, including p53, Cdc25B, Cdc25C, and securin. Inhibition of activations of these checkpoint-related molecules by caffeine significantly attenuated the 30-μM arsenite-induced G₂/M phase arrest by 93%. Our data suggest that the DNA damage responsive kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) play critical roles in arsenite-induced G₂/M phase arrest in aortic endothelial cells possibly via regulation of checkpoint-related signaling molecules including p53, Cdc25B, Cdc25C, and securin.

Keywords: Arsenite; ATM/ATR; P53; Cdc25B; Cdc25C; Securin; Mitosis

Clinical evidence for lead-induced inhibition of nitric oxide formation by Fernando Barbosa Jr; Jonas T. C. Sertorio; Raquel F. Gerlach; Jose E. Tanus-Santos (pp. 811-816).

Lead exposure has been associated with increased cardiovascular risk, which may result, at least in part, from lead-induced increases in oxidative stress and depressed nitric oxide (NO) availability. However, no previous clinical study has examined whether lead exposure is associated with significant effects on biomarkers of NO activity (plasma nitrites, nitrates, and cyclic guanosine 3′,5′-monophosphate; cGMP). We investigated whether there is an association between the circulating concentrations of nitrites, nitrates, and cGMP and the concentrations of lead in whole blood (B-Pb) or plasma (P-Pb) from 62 lead-exposed subjects (30 men and 32 women). P-Pb was determined by inductively coupled plasma mass spectrometry and B-Pb by graphite furnace atomic absorption spectrometry. Plasma nitrite and nitrate concentrations were measured using an ozone-based chemiluminescence assay. Plasma cGMP concentrations were measured using a commercial enzyme immunoassay. We found a negative correlation between plasma nitrite and B-Pb concentrations (r = −0.358; P = 0.004), and between plasma nitrite and P-Pb concentrations (r = −0.264; P = 0.038), thus suggesting increased inhibition of NO formation with increasing B-Pb or P-Pb concentrations. However, no significant correlations were found between plasma nitrate or cGMP and B-Pb or P-Pb concentrations (all P > 0.05). These findings suggest a significant inhibitory effect of lead exposure on NO formation and provide clinical evidence for a biological mechanism possibly involved the association between lead exposure and increased cardiovascular risk.

Keywords: Cyclic GMP; Lead toxicology; Nitric oxide; Nitrates; Nitrites; Plasma lead; Whole blood lead

Acrylamide exposure via the diet: influence of fasting on urinary mercapturic acid metabolite excretion in humans by Melanie I. Boettcher; Hermann M. Bolt; Jürgen Angerer (pp. 817-819).

Acrylamide (AA) is carcinogenic in animals and classified by the International Agency for Research on Cancer as probably carcinogenic in humans. Regarding the AA contents of food the diet significantly contributes to the overall AA burden of the general population. However, it is unclear to which degree the diet, apart from smoking, contributes to the internal AA exposure. Therefore the influence of an AA-free diet on the excretion of urinary mercapturic acid metabolites derived from AA in three healthy volunteers fasting for 48 h was examined. Urinary AA mercapturic acid metabolites were considerably reduced after 48 h of fasting. The levels were even well below the median level in non-smokers. This confirms that the diet is the main source of environmental AA exposure in humans, apart from smoking. Other possible AA sources could be of minor quantitative importance only.

Keywords: Acrylamide (AA); Glycidamide (GA); Diet; Human urine; Mercapturic acids

Metabolism of acrylamide to glycidamide and their cytotoxicity in isolated rat hepatocytes: protective effects of GSH precursors by Hideo Kurebayashi; Yasuo Ohno (pp. 820-828).

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxicity and prevention. Metabolism and cytotoxicity of AA and its epoxide glycidamide (GA) were studied in the hepatocytes freshly isolated from male Sprague–Dawley rats. The isolated hepatocytes metabolized AA to GA. The formation of GA followed Michaelis-Menten kinetic parameters yielded apparent K _m = 0.477 ± 0.100 and 0.263 ± 0.016 mM, V _max = 6.5 ± 2.1 and 26.4 ± 3.0 nmol/h/10⁶ cells, and CL_int = 14 ± 5 and 100 ± 12 μl/h/10⁶ cells for the hepatocytes from untreated and acetone-treated rats, respectively. There were lower K _m and marked increases in V _max (fourfold) and in CL_int (sevenfold) in acetone-treated rat hepatocytes. The data suggest that CYP2E1 played a major role in metabolizing AA to more toxic GA. Both AA and GA induced a concentration- and time-dependent glutathione (GSH) depletion of the hepatocytes. From decreasing rates of GSH contents in hepatocytes, the parameters of glutathione S-transferase (GST) in hepatocytes to AA and GA were calculated to be K _m = 1.4 and 1.5 mM, V _max = 21 and 33 nmol/h/10⁶ cells, and CL_int = 15 and 23 μl/h/10⁶ cells, respectively. GA 1.5-times more readily depleted GSH content than AA. GA decreased the viability of hepatocytes at 3 mM, but AA did not. These data indicate that GA is more toxic than AA as assessed by intracellular GSH depletion and loss of viability of hepatocytes. GSH precursors such as N-acetylcysteine and methionine provided significant anti-cytotoxic effects on the decrease of GSH content and cell viability of hepatocytes induced by GA and AA.

Keywords: Isolated hepatocytes; Acrylamide; Glycidamide; CYP2E1; Cytotoxicity; GSH precursor

Thrombin-like effect of an important green pit viper toxin, albolabrin: a bioinformatic study by Viroj Wiwanitkit (pp. 829-832).

The green pit viper venom has a major effect on the hematological system. Clinical features of venomous snakebites vary from asymptomatic to fatal bleeding. The venom is found to have a thrombin-like effect in vitro. Here, the author performs a bioinformatic analysis on the green pit viper venom focusing on its thrombin-like effect. Sequence comparison between green pit viper venom, albolabrin and thrombin was performed. In addition, the author performed a search for other human proteins closely relating to the thrombin and created a multiple sequence alignment phylogenetic tree to present the family tree of the thrombin, albolabrin and those proteins recorded in the genomic database. In conclusion, the comparative sequence analysis between green pit viper venom and thrombin gives several identities. The reported relationship on the phylogenetic tree can match with the reported in vivo function of green pit viper. Explanations on the effect of green pit viper toxin on the hemostasis can be derived from this study. Furthermore, future researches based on the reported identities can be expected.

Keywords: Bioinformatic; Green pit viper; Phylogenetic; Sequence; Structure; Albolabrin

Effects of organic chemicals derived from ambient particulate matter on lung inflammation related to lipopolysaccharide by Ken-ichiro Inoue; Hirohisa Takano; Rie Yanagisawa; Seishiro Hirano; Takahiro Kobayashi; Takamichi Ichinose; Toshikazu Yoshikawa (pp. 833-838).

The effects of components of ambient particulate matter (PM) on individuals with predisposing respiratory disorders are not well defined. We have previously demonstrated that airway exposure to diesel exhaust particles (DEP) or organic chemicals (OC) extracted from DEP (DEP–OC) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide, LPS). The present study aimed to examine the effects of airway exposure to OC extracted from urban PM (PM–OC) on lung inflammation related to LPS. ICR mice were divided into four experimental groups that intratracheally received vehicle, LPS (2.5 mg/kg), PM–OC (4 mg/kg), or PM–OC + LPS. Lung inflammation, lung water content, and lung expression of cytokines were evaluated 24 h after intratracheal administration. LPS challenge elicited lung inflammation evidenced by cellular profiles of bronchoalveolar lavage fluid and lung histology, which was further aggravated by the combined challenge with PM–OC. The combination with PM–OC and LPS did not significantly exaggerate LPS-elicited pulmonary edema. LPS instillation induced elevated lung expression of interleukin-1β, macrophage inflammatory protein-1α, macrophage chemoattractant protein-1, and keratinocyte chemoattractant, whereas the combined challenge with PM–OC did not influence these levels. All the results were consistent with our previous reports on DEP–OC. These results suggest that the extracted organic chemicals from PM exacerbate infectious lung inflammation. The mechanisms underlying the enhancing effects are not mediated via the enhanced local expression of proinflammatory cytokines.

Keywords: Ambient particulate matter; Infectious lung inflammation; Neutrophils; Cytokines

Hormonal activity of combinations of genistein, bisphenol A and 17β-estradiol in the female Wistar rat by Simone Schmidt; Gisela H. Degen; Jan Seibel; Torsten Hertrampf; Günter Vollmer; Patrick Diel (pp. 839-845).

Phytoestrogens have been described as weak estrogens,selective estrogen receptor mediators (SERMs) or to exhibit antiestrogenic properties. However, information about their activity in combination with xenoestrogens and 17β-estradiol in vivo, is limited. Therefore, the combinatory activity of the phytoestrogen genistein (Gen), the industrial chemical bisphenol A (BPA), and ethinylestradiol (EE) in ovariectomized Wistar rats was analyzed in this study. All compounds were administered orally on three consecutive days (EE at 30 μg, Gen at 100 mg and BPA at 200 mg per kg body weight per day). The pure antiestrogen fulvestrant (3 mg/kg) served as estrogen receptor (ER) antagonist control. Effects on uterine wet weight, height of the uterine epithelium, uterine clusterin (Clu) and complement C3 expression, and the height of the vaginal epithelium were examined. Treatment with Gen alone resulted in a moderate stimulation of uterine weight; in the vagina the height of the epithelium was strongly stimulated. BPA did not stimulate any of the above-mentioned parameters significantly. In combination with EE, Gen acted on most of the analyzed parameters in an additive manner, whereas BPA significantly antagonized the effects of EE on the uterine epithelium and uterine Clu expression. Given in combination with Gen, BPA was also able to antagonize the stimulatory effect of Gen on the uterine epithelium. In summary, our results demonstrate that Gen, in contrast to BPA, does not exhibit any antiestrogenic properties, even if given at high concentrations. The results of this study characterize BPA as a functional antiestrogen, very likely the result of a lack of ability to activate ER-mediated transactivation after binding to the receptor. This is not the case for Gen. Our results point to the involvement of complex molecular mechanisms in the action of Gen. These mechanisms, especially the role of ERβ have to be characterized in further investigations.

Keywords: Bisphenol A; Phytoestrogens; Genistein; Combinatory action

Avian transgenerational reproductive toxicity test with in ovo exposure by Ryo Kamata; Shinji Takahashi; Akira Shimizu; Fujio Shiraishi (pp. 846-856).

Ecological risk assessment of environmental pollutants requires effective laboratory assays and extrapolation of the resultant data to wild species. Because avian reproductive disorder and accumulation of persistent compounds in wild birds and their eggs have long been observed in polluted regions, we have developed an assay for investigating whether pollutants accumulated in eggs impair the reproduction of the exposed birds and the survival of the next generation using the Japanese quail. A typical estrogenic compound, diethylstilbestrol (DES), dissolved in olive oil was injected into the air-chamber of fertilized eggs on day 10 of incubation. After sexual maturation of hatched chicks, we made pairs of male and female quails following an observation period of egg production and colleted their eggs. The collected eggs were incubated and checked for the fertility and hatchability, and then the hatchlings were raised and observed in growth for 3 weeks. A dosage of 5 ng/g per egg of DES caused eggshell thinning in eggs laid by exposed females and reduction in eggshell strength. DES also induced shortening of the left oviduct and unexpected development of the right oviduct, while testis weight was reduced symmetrically. The ability of quail pairs to produce offspring was significantly diminished by exposure of females to DES independently of exposure of males, which mainly arose from production of abnormal and inviable eggs. Fertility of normal-shelled eggs and hatchability of fertilized eggs were unchanged regardless of treatments. External morphological abnormalities, which were mostly unopened toes of the foot, were frequently observed in hatchlings from exposed males independently of exposure of females. Additionally, we attempted to extrapolate the experimental results to the northern bobwhite and to predict population trends for quails in a polluted habitat using a population projection model composed of a combination of a Leslie matrix and the logistic equation. In the event of accumulation of an estrogenic compound equivalent to a dosage of 5 ng/g DES in quail eggs, the average population size was predicted to decrease by 20.2% after 1 year, to approximately half after 4 years, and to a fifth after 14 years. When observed weakening of individuals and the risk of egg breakage are taken into consideration, the decline in population was further accelerated. The proposed assay appears to be suitable not only for assessing adverse effects of chemicals on avian reproduction but for population projection of affected wild birds.

Keywords: Avian reproductive disorder; Japanese quail; In ovo exposure; Population projection; Diethylstilbestrol

In ovo exposure quail assay for risk assessment of endocrine disrupting chemicals by Ryo Kamata; Shinji Takahashi; Akira Shimizu; Masatoshi Morita; Fujio Shiraishi (pp. 857-867).

Although there are in vivo assays using various organisms for the risk assessment of chemicals with endocrine disrupting properties, effective experimental methods for avian species are still under debate. We have developed an in ovo exposure assay using Japanese quail eggs, aimed at assessing disrupting effects on avian reproductive development and function. Hybrid eggs from Brazilian Brown male and White Egg female quails, which can be genetically sexed by their plumage color after hatching, were prepared, and test materials dissolved in olive oil were injected into the air-chamber on day 10 of incubation. After sexual maturation of hatched chicks, we observed egg production by females and the egg quality and male-typical reproductive behavior, and then examined reproductive system morphology and serum steroid concentrations in both sexes. Treatment with a synthetic estrogen, diethylstilbestrol (DES, 0.5–50 ng/g egg), dose-dependently reduced the eggshell thickness and strength of eggs. A few females treated with 5 ng/g DES per egg produced soft-shelled/unmarked eggs, and all laying females treated with 50 ng/g egg produced eggs completely lacking shells. DES also induced shortening of the left oviduct and abnormal development of the right oviduct in a dose-dependent manner, while testis weight was reduced symmetrically. In addition, 2,2′,4′,6′-tetrachlorobiphenyl-4-ol (10–1,000 ng/g egg), which previously showed relatively high estrogenic activity in vitro, caused dose-dependent shortening of the left oviduct and reduction in testis weight. The methods for evaluating endocrine disrupting effects and preparing experimental birds proposed in the present study are expected to facilitate assays for avian reproductive toxicology.

Keywords: Avian reproductive disorder; Japanese quail; In ovo exposure; Eggshell thinning; Diethylstilbestrol; 2,2′,4′,6′-Tetrachlorobiphenyl-4-ol

In vivo micronucleus test in mice with 1-phenylethanol by Günter Engelhardt (pp. 868-872).

1-Phenylethanol is one of the major primary phase-I metabolites of ethylbenzene. In principle it may yield an electrophilic intermediate by phase-II metabolism. Because of the extensive use of ethylbenzene as a solvent, 2-year carcinogenicity inhalation studies were carried out leading to renal hyperplasia and tubular neoplasms both in male and female rats and alveolar/bronchiolar neoplasms in male mice and hepatocellular neoplasms in female mice. Whereas the mechanism underlying the increased renal tumor incidences in rats has been clarified, the mechanism of tumor formation (genotoxic or nongenotoxic mode of action) in the lung and liver of mice is still unclear. The genotoxicity data available to date for 1-phenylethanol include in vitro studies using either bacteria (Salmonella reverse mutation assay, E. coli Pol A⁺/Pol A⁻ test) or mammalian cells (mouse lymphoma assay, chromosome aberration test and sister chromatid exchanges using CHO cells). These experiments, however, did not always follow current standard procedures and some of the data obtained are compromised and not always convincing. The present database thus does not allow a definitive assessment of the in vitro genotoxic potential of 1-phenylethanol. The in vitro database suggests that clastogenicity may be the most relevant genetic end point, and therefore an in vivo micronucleus assay in mouse bone marrow was carried out. The animals were given 1-phenylethanol in single oral doses up to the maximum tolerated dose of 750 mg/kg body weight. Bone marrow was sampled 24 and 48 h after treatment. Under the experimental conditions used, there was no evidence of increased micronuclei frequencies at any dose or sampling time. These findings indicate that 1-phenylethanol is not clastogenic in vivo. This information, together with other negative or inconclusive genotoxicity data available so far, suggests a nongenotoxic mode of action responsible for the lung and liver tumors observed in mice following 2 years of inhalation exposure to ethylbenzene.

Keywords: 1-Phenylethanol; Ethylbenzene; Carcinogenicity; Genotoxicity; Micronucleus assay; Clastogenicity; Aneugenicity

Senna: a laxative devoid of carcinogenic effects by F. Borrelli; G. Aviello; R. Capasso; F. Capasso (pp. 873-873).

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Archives of Toxicology (v.80, #12)