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Archives of Toxicology (v.75, #11-12)

Health hazards due to the inhalation of amorphous silica by R. Merget; T. Bauer; H. Küpper; S. Philippou; H. Bauer; R. Breitstadt; T. Bruening (pp. 625-634).

Occupational exposure to crystalline silica dust is associated with an increased risk for pulmonary diseases such as silicosis, tuberculosis, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and lung cancer. This review summarizes the current knowledge about the health effects of amorphous (non-crystalline) forms of silica. The major problem in the assessment of health effects of amorphous silica is its contamination with crystalline silica. This applies particularly to well-documented pneumoconiosis among diatomaceous earth workers. Intentionally manufactured synthetic amorphous silicas are without contamination of crystalline silica. These synthetic forms may be classified as (1) wet process silica, (2) pyrogenic ("thermal" or "fumed") silica, and (3) chemically or physically modified silica. According to the different physico-chemical properties, the major classes of synthetic amorphous silica are used in a variety of products, e.g. as fillers in the rubber industry, in tyre compounds, as free-flow and anti-caking agents in powder materials, and as liquid carriers, particularly in the manufacture of animal feed and agrochemicals; other uses are found in toothpaste additives, paints, silicon rubber, insulation material, liquid systems in coatings, adhesives, printing inks, plastisol car undercoats, and cosmetics. Animal inhalation studies with intentionally manufactured synthetic amorphous silica showed at least partially reversible inflammation, granuloma formation and emphysema, but no progressive fibrosis of the lungs. Epidemiological studies do not support the hypothesis that amorphous silicas have any relevant potential to induce fibrosis in workers with high occupational exposure to these substances, although one study disclosed four cases with silicosis among subjects exposed to apparently non-contaminated amorphous silica. Since the data have been limited, a risk of chronic bronchitis, COPD or emphysema cannot be excluded. There is no study that allows the classification of amorphous silica with regard to its carcinogenicity in humans. Further work is necessary in order to define the effects of amorphous silica on morbidity and mortality of workers with exposure to these substances.

Keywords: Non-crystalline Amorphous Silica, Silicosis Bronchitis Emphysema Airway disease Carcinoma

A repeated 28-day oral dose toxicity study of 17α-methyltestosterone in rats, based on the 'Enhanced OECD Test Guideline 407' for screening the endocrine-disrupting chemicals by Kazushi Okazaki; Takayoshi Imazawa; Hideaki Nakamura; Fumio Furukawa; Akiyoshi Nishikawa; Masao Hirose (pp. 635-642).

As part of the international validation project to establish the Enhanced OECD Test Guideline 407, we performed a 28-day repeated-dose toxicity study of 17α-methyltestosterone, an exogenous androgen agonist. Special attention was paid to the sensitivity of additional parameters for detecting endocrine-related effects of endocrine-disrupting chemicals, based on the existing Test Guideline 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 17α-methyltestosterone was administered daily by gavage at doses of 0 (control), 5, 20 and 80 mg/kg body weight per day. Male rats were killed on the day after the 28th administration and females on the day of the diestrus stage during the 4 day period after the 28th administration. Male rats receiving 80 mg/kg 17α-methyltestosterone demonstrated decreases in testis and epididymis weights, atrophy of seminiferous tubules and Leydig cells, and degenerated pachytene spermatocytes in the testes and degenerated germ cells in the epididymides as major alterations. Female rats showed abnormal estrous cycles, decreases in ovary and adrenal weights, increase in immature follicles with decreased corpus lutea in the ovaries at doses of 5 mg/kg and higher, as well as atrophy of zona reticularis in the adrenals and increase in mammary gland secretion at 20 mg/kg and above. Dilatation of the lumina and apoptosis of endometrial cells in the uterus, mucinification in the vagina and increase in serum follicle-stimulating hormone were seen with 80 mg/kg. Among the parameters examined in the present experimental system, effects of 17α-methyltestosterone on endocrine-related organs were detected in organ weights and histopathological examination of both sexes, and in serum hormones and estrous cycle of females. Based on these results, the no-observed-adverse-effect level (NOAEL) in the present study was estimated to be below 5 mg/kg per day. In particular, effects were most sensitively detected by organ weights and histopathological examination of sexual organs.

Keywords: 17α-Methyltestosterone Androgen Rat Enhanced OECD Test Guideline 407 Endocrine disrupters

Toxicokinetic interaction of 2,5-hexanedione and methyl ethyl ketone by Rong Yu; Dale Hattis; Elliot M. Landaw; John R. Froines (pp. 643-652).

Co-exposure to methyl ethyl ketone (MEK) potentiates the neurotoxicity of n-hexane in humans as well as in animals. This effect is associated with increased persistence of 2,5-hexanedione (2,5-HD) in blood, probably due to inhibition of 2,5-HD phase II biotransformation by MEK. There is no previous quantitative toxicokinetic model to describe this interaction. In this study we constructed a toxicokinetic model to depict the inhibition of 2,5-HD metabolism and elimination by MEK. Experimental data on 2,5-HD blood concentrations in rats from a published study were used to estimate model parameters. Three different inhibition mechanisms were evaluated: competitive, uncompetitive, and noncompetitive inhibition. Extrapolation from high to low doses was made to assess the interactive effects of MEK on 2,5-HD beyond experimental conditions. The models developed successfully described the toxicokinetic behavior of 2,5-HD when inhibited by MEK. The competitive inhibition model yielded a much lower estimate for the constant (65.5 mg/l) of 2,5-HD inhibition by MEK than did the uncompetitive and noncompetitive models (403 and 440 mg/l, respectively). The apparent half-life of 2,5-HD appeared to be a linear function of the Michaelis-Menten constant, and 2,5-HD and MEK concentrations in rats. The area under the curve of 2,5-HD in blood of rats was a nonlinear function of 2,5-HD and MEK concentrations in the blood. This study highlights the importance of the interactive effect of MEK on deactivation and elimination of 2,5-HD, and further illustrates the advantage of toxicokinetic modeling to investigate chemical interactions associated with exposure to multiple chemical agents.

Keywords: Chemical mixture Toxicokinetic interaction Metabolic inhibition 2,5-Hexanedione Methyl ethyl ketone

Tissue concentrations and induction of a hepatic monooxygenase in male Wistar rats after repeated doses of defined polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDDs and PCDFs) mixtures by Wolfgang Körner; Georg Golor; Thomas Schulz; Thomas Wiesmüller; Hanspaul Hagenmaier; Diether Neubert (pp. 653-664).

Two groups of male Wistar rats were treated 16 times (every 3rd day) subcutaneously with a defined mixture of polychlorinated dibenzo-p-dioxins (PCDDs) or of polychlorinated dibenzofurans (PCDFs). These mixtures contained no measurable amount of 2,3,7,8-TCDD. Each single dose was calculated to contain either 57 ng I-TEq (international 2,3,7,8-T4CDD toxicity equivalencies)/kg body weight of the PCDD mixture or 39 ng I-TEq/kg body weight of the PCDF mixture. Both mixtures contained a large excess of non-2,3,7,8-substituted congeners. The activities of ethoxyresorufin O-deethylase (EROD) in liver microsomes were correlated with the corresponding concentrations of PCDDs or PCDFs in hepatic tissue. Data were compared with results obtained after single injections of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-T4CDD). As expected, a complex kinetic situation resulted, because of the different tissue distributions and elimination half-lives of the various congeners: (1) 2,3,7,8-substituted PCDDs: the time course of the concentrations in liver and adipose tissue was similar for all congeners, the levels increased during the treatment period and decreased after treatment. Tissue concentrations of all 2,3,7,8-substituted PCDDs were considerably higher in liver than in adipose tissue. The liver/adipose tissue concentration ratios increased with the degree of chlorination. The ratio of 1,2,3,7,8-P5CDD was much lower than those of all other 2,3,7,8-substituted congeners. (2) 2,3,7,8-substituted PCDFs: 1,2,3,7,8-P5CDF was rapidly eliminated from liver and adipose tissue while 2,3,4,7,8-P5CDF largely persisted after the treatment period in both tissues. 2,3,7,8-T4CDF was eliminated even more rapidly than 1,2,3,7,8-P5CDF and could not be detected after treatment in both tissues. Time courses of the concentrations of 2,3,4,7,8-P5CDF, H6CDFs, H7CDFs and OCDF in liver and adipose tissue were similar: the levels of all congeners increased during the treatment period but no clear-cut decrease was observed within 34 days after the last treatment. Tissue concentrations of all 2,3,7,8-substituted PCDFs were higher in liver than in adipose tissue. The liver/adipose tissue concentration ratios increased with the degree of chlorination. The ratios of 2,3,7,8-T4CDF and 1,2,3,7,8-P5CDF were much lower than those of all other 2,3,7,8-substituted congeners. (3) non-2,3,7,8-substituted PCDDs and PCDFs: a number of non-2,3,7,8-substituted PCDD and PCDF congeners were found in both tissues in concentrations below 1 ng/g. In adipose tissue nearly all congeners were found during the treatment period showing a decrease after the treatment. In liver samples, many higher chlorinated PCDF congeners (with >4 chlorine atoms) could be detected. Most of those substituted in three of the four 2, 3, 7 and 8-positions persisted after treatment. In contrast, only one 1,4,6,9-substituted isomer of each PCDD homologue group was found during treatment with high recoveries after the third injection, but a rapid decline occurred already during the treatment period. (4) EROD activity: a good linear relationship (when using a double-log plot) between the EROD activities and the hepatic concentrations (ng I-TEq/g tissue) was found both in the PCDD-treated (r 2=85.8%) and in the PCDF-treated group (r ²=87.3%). A similar correlation (r 2=95.6%) was observed in rats treated with 2,3,7,8-TCDD alone (concentration range in liver tissue: 0.2 to 9.7 ng/g wet weight). The concentration-response curves for both the PCDD and PCDF mixtures run parallel to the curve for 2,3,7,8-T4CDD. However, the inductive potency of the PCDD or PCDF mixture was approximately 3-fold or 4-fold lower, respectively, compared with the inductive potency of 2,3,7,8-T4CDD. Thus, the I-TE factors overestimated the potency of the mixtures in the concentration range tested and taking EROD induction as an end point.

Keywords: Ethoxyresorufin O-deethylase Kinetics Polychlorinated dibenzofurans Polychlorinated dibenzo-p-dioxins Tissue distribution Toxicity equivalency factors

Metabolism and CYP-inducer properties of astaxanthin in man and primary human hepatocytes by A. Kistler; H. Liechti; L. Pichard; E. Wolz; G. Oesterhelt; A. Hayes; P. Maurel (pp. 665-675).

Previous investigations in the rat have shown that the non-provitamin A carotenoid astaxanthin is metabolized into 3-hydroxy-4-oxo-β-ionone and 3-hydroxy-4-oxo-7,8-dihydro-β-ionone and, in addition, is a potent CYP1A gene inducer. Here we investigated the metabolism of this compound as well as its capacity to induce CYP genes in primary cultures of human hepatocytes. Free metabolites of ¹⁴C-astaxanthin produced in this cellular model were purified by high pressure liquid chromatography (HPLC) and identified by gas chromatography–mass spectrometry (GC-MS) analyses as 3-hydroxy-4-oxo-β-ionol and 3-hydroxy-4-oxo-β-ionone. In addition, deconjugation of polar compounds by glusulase and further analyses with HPLC and GC-MS revealed four radiolabeled metabolites including: 3-hydroxy-4-oxo-β-ionol, 3-hydroxy-4-oxo-β-ionone, and their reduced forms, 3-hydroxy-4-oxo-7,8-dihydro-β-ionol and 3-hydroxy-4-oxo-7,8-dihydro-β-ionone. The same four metabolites were identified in human plasma from two volunteers who had orally taken 100 mg astaxanthin 24 h before blood collection. In cultured hepatocytes, astaxanthin was a significant inducer of the major cytochrome P450 enzyme, CYP3A4 as well as of CYP2B6, but not of other CYPs, including those from CYP1A and CYP2C families. The lack of autoinduction of astaxanthin metabolism in human hepatocytes suggests that neither CYP3A4 nor CYP2B6 contribute to the formation of metabolites. We conclude that metabolism of astaxanthin and its CYP-inducing capacity are different in humans and in rats. The novel methodology used in our studies could be extended to evaluating the role of metabolites of more important carotenoids such as β-carotene in differentiation and carcinogenicity.

Keywords: Carotenoids Astaxanthin Metabolism Human Cultured hepatocytes

Age-related effects of chlorpyrifos on muscarinic receptor-mediated signaling in rat cortex by Hengshan Zhang; Jing Liu; Carey N. Pope (pp. 676-684).

Chlorpyrifos (CPF) is a widely used organophosphorus pesticide. Earlier work from our laboratory and others has demonstrated that the sensitivity to CPF exposure changes markedly during maturation. A number of studies suggest that in addition to inhibiting acetylcholinesterase (AChE), CPF oxon may also interact directly with m2 and/or m4 subtypes of muscarinic acetylcholine receptors (mAChRs). In the present study, we investigated the in vivo effects of CPF exposure on phosphoinositide (PI) hydrolysis and cAMP formation, second-messenger systems coupled to m1, m3 and m5 (PI hydrolysis) or m2 and m4 (cAMP formation) mAChRs. Neonatal (7-day), juvenile (21-day) and adult (90-day) rats were treated with either peanut oil s.c. or CPF s.c. at 0.3× or 1× the maximum tolerated dosage (MTD: 45, 127 and 279 mg/kg for 7-day, 21-day and 90-day rats, respectively). Neurochemical end-points including AChE activity, muscarinic receptor ([³H]quinuclidinyl benzilate, and [³H]oxotremorine) binding, PI hydrolysis, and cAMP formation in cortex were evaluated at 4 h, 24 h, or 96 h after treatment. Under these conditions, relatively similar maximal degrees of cholinesterase (ChE) inhibition were noted, but times to peak inhibition varied among these age groups (24 h in neonates and juveniles, 96 h in adults). Total muscarinic receptor (QNB) binding was reduced in all three age groups with 1× MTD exposure, at both 24 h and 96 h in neonates and juveniles, but only at 96 h in adults. Oxotremorine binding was also reduced at 96 h after MTD exposure in all three age groups. Neither basal nor carbachol-stimulated IP accumulation was affected in any age group or at any time point following CPF exposure. In contrast, basal cAMP formation was significantly increased by MTD exposure in all three age groups 4 h after exposure, and at 4 h, 24 h, and 96 h after exposure in juveniles. Forskolin/Mn²⁺-stimulated cAMP formation was increased in neonates and juveniles at 96 h, and in juveniles also at 24 h, but was significantly decreased in adults at 96 h after MTD exposure. Oxotremorine-mediated inhibition of cAMP formation was significantly greater at 96 h after MTD exposure in all three age groups. These results provide further evidence that the cortical cAMP signaling pathway may be particularly sensitive to CPF exposure in neonatal, juvenile, and adult rats, possibly due to a direct interaction between CPF (or its oxon) and mAChRs or other components of the adenylyl cyclase cascade.

Keywords: Organophosphates Acetylcholinesterase inhibition Phosphoinositide cAMP

The coffee components kahweol and cafestol induce γ-glutamylcysteine synthetase, the rate limiting enzyme of chemoprotective glutathione synthesis, in several organs of the rat by Wolfgang W. Huber; Gerlinde Scharf; Walter Rossmanith; Sonja Prustomersky; Bettina Grasl-Kraupp; Barbara Peter; Robert J. Turesky; Rolf Schulte-Hermann (pp. 685-694).

The coffee components kahweol and cafestol (K/C) were reported to be protective against mutagenic damage by heterocylic amines and aflatoxin B1 in the rat, while in humans the consumption of coffee with a high K/C content was associated with a lower rate of colon tumors. An important mechanism of this antimutagenic effect appears to be the potential of K/C to induce glutathione-S-transferase (GST) and to enhance hepatic levels of glutathione (GSH), the co-factor of GST, which is independently involved in further protective mechanisms. In the present study, we investigated mechanisms and organ specificities (liver, kidney, lung, colon) of the K/C effect on GSH levels, and particularly the role of γ-glutamylcysteine synthetase (GCS), the rate limiting enzyme of GSH synthesis. Chows containing one of four concentrations of either a 1:1 mixture of K/C (0.012–0.122%) or of cafestol alone (0.006–0.061%) were fed to male F344 rats for 10 days. In the K/C-treated livers, a dose-dependent increase of up to 2.4-fold in the activity of GCS was observed, being statistically significant even at the lowest dose, and associated with an increase in GSH of up to three-fold. Notably, the highest dose doubled the hepatic mRNAs of the heavy and light subunits of GCS, suggesting enhanced transcription. In the extrahepatic organs, GCS activity and GSH levels were increased as well, although more moderately than in the liver. Since enhancement of GCS had also been observed as a consequence of oxidative stress, the possibility of such an involvement in the actions of K/C was examined by determining hepatic thiobarbituric acid reactive substances and the ratio of oxidized and reduced GSH. However, no evidence of oxidative stress was detected. In summary, K/C increased GSH levels apparently through the induction of the rate limiting enzyme of GSH synthesis, which may be a key factor in the chemopreventive potential of coffee components.

Keywords: Coffee Chemoprevention Glutathione γ-Glutamylcysteine synthetase Rat

Novel action of lignans isolated from Hernandia nymphaeifolia on Ca²⁺ signaling in human neutrophils by Yu-Ying Chao; Warren Su; Chung-Ren Jan; Ying-Chin Ko; Jih-Jung Chen; Jin-Shiung Cheng; Chun-Peng Liu; Yuk-Keung Lo; Kang-Ju Chou; Kam-Chung Lee; Wei-Chung Chen; Ih-Sheng Chen (pp. 695-702).

The effects of five lignans (epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin, yatein) isolated from Hernandia nymphaeifolia (Presl.) Kubitzki (Hernandiaceae) on intracellular Ca²⁺ levels ([Ca²⁺]_i) in human neutrophils were investigated by using fura-2 as a fluorescent probe. In both Ca²⁺-containing and Ca²⁺-free media, the lignans (50–100 µM) did not alter basal [Ca²⁺]_i but inhibited the [Ca²⁺]_i increase induced by platelet activating factor (PAF, 10 µM), leukotriene B₄ (LTB₄, 0.2 µM), and thapsigargin (1 µM) to different extents. In Ca²⁺-free medium, after depleting stores of Ca²⁺ with PAF, LTB₄ or thapsigargin, addition of 3 mM Ca²⁺ induced Ca²⁺ influx. Each of the lignans (50–100 µM) caused 39–89% inhibition of PAF-induced Ca²⁺ influx; whereas only epi-aschantin was able to inhibit LTB₄- and thapsigargin-induced Ca²⁺ influx by 54–79%. Together, the results suggest that in human neutrophils, these lignans did not alter basal [Ca²⁺]_i but inhibited Ca²⁺ movement induced by Ca²⁺ mobilizing agents.

Keywords: Calcium mobilization Calcium signaling Lignans Neutrophils Human

Uterotrophic and Hershberger assays for n-butylbenzene in rats by Kanji Yamasaki; Masakuni Sawaki; Shuji Noda; Mineo Takatsuki (pp. 703-706).

We performed the uterotrophic and Hershberger assays proposed by the OECD to investigate the estrogenic and androgenic effects of n-butylbenzene (nBB). For the uterotrophic assay, nBB was injected subcutaneously at doses of 0, 40, 200, 1000 and 2000 mg/kg to 19-day-old rats for 3 days. In some rats, ethynylestradiol (EE) was also injected subcutaneously at a dose of 0.6 µg/kg after the administration of nBB. There were essentially no differences in the uterine wet or blotted weights between the control and any of the nBB-treated groups, or between the control and any of the nBB plus EE-treated groups. For the Hershberger assay, nBB was administered orally at doses of 0, 200 and 600 mg/kg to 56-day-old castrated rats for 10 days. In some rats, 0.4 mg/kg testosterone propionate (TP) was also administered by subcutaneous injection after the administration of nBB. Doses of 0, 200 and 600 mg/kg nBB were also administered orally to non-castrated rats. The weights of the accessory sex organs of the castrated rats showed no significant differences between the control and any of the nBB-treated groups or between the control and the nBB plus TP-treated groups. No significant differences in the weights of the accessory sex organs of the non-castrated rats were observed between the control and the nBB-treated groups. These findings suggest that nBB does not have any endocrine-disrupting properties on in vivo screening tests.

Keywords: n-Butylbenzene Uterotrophic assay Hershberger assay Ethynylestradiol Testosterone propionate

Steroid hormone progesterone induces cell proliferation and abnormal mitotic processes in rat liver by Luis D. Boada; Manuel Zumbado; Isidoro del Río; Alfonso Blanco; Santiago Torres; José G. Monterde; Juan L. Afonso; Juan J. Cabrera; Bonifacio N. Díaz-Chico (pp. 707-716).

The aim of this study was to evaluate the acute hepatic effects exerted by the steroid hormone progesterone (PR) in the rat. Although the liver is not a target tissue for this hormone, a number of hepatic actions of PR have been described, and, furthermore, a specific binding site for PR (PBS) exists in rat liver microsomes. Immature male rats were treated intraperitoneally with 60 mg/kg PR per day for 1, 5 or 10 days, and different parameters were evaluated in order to detect possible alterations in liver cells. Morphological study of the livers did not present images of cytotoxicity in any group of animals. The presence of a clear hyperplasia of smooth endoplasmic reticulum (SER) was noteworthy, mainly seen in perilobular hepatocytes. Despite this SER increase, the levels of cytochrome P450 (Cyt P450) significantly decreased after 10 days of PR administration. Similarly, the concentration of PBS was significantly decreased after 10 days of treatment with PR. On the other hand, these studies revealed a clear increase of mitotic activity and Ki-67 labelling index in the livers of animals treated with PR; furthermore, livers of PR-treated animals showed an increased percentage of binucleate hepatocytes. Flow cytometry analysis showed that although ploidy status of liver cells was not modified in any case the percentage of diploid nuclei in S-phase decreased during treatment with PR. The most relevant finding was the presence of abnormal mitosis and c-mitosis in livers from animals from all PR-treated groups. This study demonstrates that PR (a) does not induce cytotoxicity although it can induce cell proliferation and spindle disturbances in liver cells, (b) may also modulate the drug-metabolizing liver enzyme function, and (c) downregulates the expression of its own microsomal specific binding site.

Keywords: Progesterone Liver Cytochrome P450 Cell proliferation c-Mitosis Ploidy

Embryonic exposure to lead: comparison of immune and cellular responses in unchallenged and virally stressed chickens by Ji-Eun Lee; Syed A. Naqi; Elizabeth Kao; Rodney R. Dietert (pp. 717-724).

Lead, a ubiquitous environmental contaminant, has been shown to modulate various functions of the immune system and decrease host resistance to infectious disease. However, limited information is available concerning the direct effects of lead on the host immune response to an infectious agent after developmental exposure. The current study utilized chickens to examine the effect of embryonic lead exposure on immune and cellular responses during viral challenge. Sublethal doses of lead were introduced into fertilized Cornell K Strain White Leghorn chicken eggs via the air sac at day 5 or day 12 of embryonic development (designated as E5 and E12, respectively). Four-week-old female chickens were inoculated with infectious bronchitis virus (IBV) strain M41. Antibody titer to IBV, delayed-type hypersensitivity (DTH) response against bovine serum albumin (BSA), the absolute number and percentage of leukocyte subpopulations, and interferon-γ (IFN-γ)-like cytokine production by splenocytes were evaluated at 5–6 weeks of age. While antibody response to IBV in juvenile chicks was unaffected by the in ovo lead exposure, IFN-γ-like cytokine production by splenocytes was significantly depressed following lead exposure at both developmental stages. In contrast with this pattern, the DTH response against BSA was unaffected following E5 exposure, but was significantly decreased after E12 exposure to lead. These changes were similar to those previously reported in chickens not exposed to IBV. While lead exposure at E5 induced significant changes in the percentage of circulating heterophils at 1 day postinfection (dpi), lead did not cause any change in relative leukocyte counts after E12 exposure. At 7 dpi, E5 lead exposure resulted in decreased absolute number and percentage of circulating lymphocytes, while total leukocyte counts, and the absolute number and percentage of circulating monocytes and heterophils were significantly reduced in E12 lead-exposed chickens. These results suggest that low-level exposure to lead has a direct effect on the developing chicken immune system, which is evident even during a postnatal infection. Furthermore, some of the changes were observed only when chicks were stressed by the viral infection. It appears that lead exposure during different stages of embryonic development is likely to result in different immunotoxic outcomes in juveniles.

Keywords: Lead Infectious bronchitis virus Delayed-type hypersensitivity (DTH) IFN-γ production Leukocyte subpopulations

Ultrastructural characterization of murine limb buds after in vitro exposure to grepafloxacin and other fluoroquinolones by Mehdi Shakibaei; Irmela Baumann-Wilschke; Marcus Rücker; Ralf Stahlmann (pp. 725-733).

The effects of selected quinolones (levofloxacin, lomefloxacin, temafloxacin and grepafloxacin) on growth and differentiation of murine limb buds were studied in vitro. Ciprofloxacin and ofloxacin served as controls. We used limb buds from 12-day-old mouse embryos that were grown for 6 days in a serum-free, standard or magnesium-deficient medium. Besides evaluation under a dissecting microscope, we used electron microscopy to characterize the effects in detail. The following results are noteworthy. (1) Comparing the effects of standard and magnesium-deficient medium after 3 and 6 days in culture, we found ultrastructural changes after 6 days only. (2) Direct comparison of ofloxacin (racemate) and levofloxacin (L-enantiomer) showed that they had a similar, rather low, potential for affecting cartilage development. (3) The effects of temafloxacin and ciprofloxacin were more pronounced in magnesium-deficient medium, but those of the other drugs were not. (4) Grepafloxacin was the most active quinolone in this assay. It impaired growth and differentiation of limb buds at 30 mg/l; at higher concentrations the explants did not grow. With lower concentrations of 10 mg grepafloxacin/l, no effects were detectable under a dissecting microscope but characteristic changes were seen by electron microscopy. We observed electron-dense aggregates on and within chondrocytes, detachment of the cell membrane from the matrix with matrix-free pericellular areas around chondrocytes, and swelling of cell organelles such as mitochondria and rough endoplasmic reticulum. (5) The affinity of grepafloxacin for divalent cations (Mg²⁺, Ca²⁺) was studied by measuring the fluorescence of grepafloxacin solution at various concentrations of Mg²⁺and Ca²⁺. Grepafloxacin showed a relatively high affinity for Ca²⁺ in the fluorescence assay, which was more pronounced than the affinities of six other fluoroquinolones tested before.

Keywords: Fluoroquinolones In vitro Magnesium Mouse Cartilage

Detection of ochratoxin A-induced DNA damage in MDCK cells by alkaline single cell gel electrophoresis (comet assay) by Stefan Lebrun; Wolfram Föllmann (pp. 734-741).

The mycotoxin ochratoxin A (OTA), a widespread contaminant of food and feedstuffs, is nephrotoxic, immunosuppressive and carcinogenic in domestic and laboratory animals. Additionally, it is suspected as being responsible for urinary tract tumours in patients suffering from Balkan endemic nephropathy. Moreover, evidence has accumulated that OTA is a genotoxic carcinogen, although the mechanism that results in DNA damage has not been fully resolved. In this study, the induction of DNA damage by OTA and the subsequent DNA repair was investigated by alkaline single cell gel electrophoresis (comet assay) in cells originally derived from the kidney, a target organ of OTA. With modifications of the method, the influence of OTA uptake into the cells and of DNA repair on the genotoxic effect of OTA should be investigated. In Madin-Darby canine kidney (MDCK) cells, OTA induced single-strand breaks in a concentration dependent manner. When an external metabolising enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By co-incubation with methotrexate or with the mycotoxin citrinin, a substrate of the organic anion transporter, the adverse effect of OTA was inhibited. When DNA repair was inhibited by addition of cytosine arabinoside and hydroxyurea, the tail length increased dramatically and all treated cells showed single-strand breaks. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the DNA damage within 2 h. Adverse effects on the mechanisms of DNA repair, or exposure to OTA in periods of reduced DNA repair capacity may influence the genotoxic potency of OTA and have to be regarded as a further mechanism by which genotoxic effects of OTA can be performed.

Keywords: Mycotoxin Ochratoxin A DNA damage DNA repair MDCK cells Alkaline single cell gel electrophoresis

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Archives of Toxicology (v.75, #11-12)