Determination of genotoxicity by the Comet assay applied to murine precision-cut lung slices.

Precision-cut lung slices (PCLSs) are an organotypic lung model that is widely used in pharmacological, physiological, and toxicological studies. Genotoxicity testing, as a pivotal part of early risk assessment, is currently established in vivo in various organs including lung, brain, or liver, and in vitro in cell lines or primary cells. The aim of the present study was to provide the three-dimensional organ culture PCLS as a new ex vivo model for determination of genotoxicity using the Comet assay. Murine PCLS were exposed to increasing concentrations of ethyl methane sulfonate 'EMS' (0.03-0.4%) and formalin (0.5-5mM). Tissue was subsequently dissociated, and DNA single-strand breaks were quantified using the Comet assay. Number of viable dissociated lung cells was between 4×10(5) and 6.7×10(5)cells/slice. Even treatment with EMS did not induce toxicity compared to untreated tissue control. As expected, DNA single-strand breaks were increased dose-dependently and significantly after exposure to EMS. Here, tail length rose from 24µm to 75µm. In contrast, formalin resulted in a significant induction of DNA cross-links. The effects induced by EMS and formalin demonstrate the usefulness of PCLS as a new ex vivo lung model for genotoxicity testing in the early risk assessment of airborne substances in the future.

Response, thermal regulatory threshold and thermal breakdown threshold of restrained RF-exposed mice at 905 MHz.

The objective of this study was the determination of the thermal regulatory and the thermal breakdown thresholds for in-tube restrained B6C3F1 and NMRI mice exposed to radiofrequency electromagnetic fields at 905 MHz. Different levels of the whole-body averaged specific absorption rate (SAR = 0, 2, 5, 7.2, 10, 12.6 and 20 W kg(-1)) have been applied to the mice inside the 'Ferris Wheel' exposure setup at 22 +/- 2 degrees C and 30-70% humidity. The thermal responses were assessed by measurement of the rectal temperature prior, during and after the 2 h exposure session. For B6C3F1 mice, the thermal response was examined for three different weight groups (20 g, 24 g, 29 g), both genders and for pregnant mice. Additionally, NMRI mice with a weight of 36 g were investigated for an interstrain comparison. The thermal regulatory threshold of in-tube restrained mice was found at SAR levels between 2 W kg(-1) and 5 W kg(-1), whereas the breakdown of regulation was determined at 10.1 +/- 4.0 W kg(-1)(K = 2) for B6C3F1 mice and 7.7 +/- 1.6 W kg(-1)(K = 2) for NMRI mice. Based on a simplified power balance equation, the thresholds show a clear dependence upon the metabolic rate and weight. NMRI mice were more sensitive to thermal stress and respond at lower SAR values with regulation and breakdown. The presented data suggest that the thermal breakdown for in-tube restrained mice, whole-body exposed to radiofrequency fields, may occur at SAR levels of 6 W kg(-1)(K = 2) at laboratory conditions.

Cross-fostering inhalation toxicity study with HCFC-123 in lactating Sprague-Dawley rats.

A study was performed in Sprague-Dawley rats (Crl:CD BR) to differentiate between effects of hydrofluorocarbon 123 (HCFC-123) on the lactating dam or on the fetus using fostering and cross-fostering of the offspring. Pregnant and/or lactating dams without the pups present were exposed to the test substance (1000 ppm) or clean air by whole-body inhalation for 6 h/day from day 6 to 19 post conceptionem (p.c.) and from day 5 to 21 post partum (p.p.). Pups were cross-fostered to new dams within the first 2 days after birth. Treatment of the mothers with HCFC-123 led to decreases in serum glucose, cholesterol, and triglycerides and increases in absolute and relative maternal liver weights. Decreased litter and individual pup weight and decreased serum triglycerides were observed in the pups of treated foster mothers. Treatment of the mothers with HCFC-123 did not influence milk production based on the body weight difference of the dam before suckling and 60 min after beginning of suckling using 12-pup "standard litters" of untreated dams. Total fat, glucose, and protein contents in the milk were also not influenced by the treatment. Trifluoroacetic acid (TFA), a main metabolite of HCFC-123, was observed in urine samples of standard litters that had been nursed by treated dams. In conclusion, the effects on offspring due to HCFC-123 treatment consisted of decreased pup weight and decreased serum triglycerides at weaning. All effects were due to treatment of the lactating dams, as no prenatally induced effects were found. Since milk production and nutritional constituents of the milk were not influenced, but significant amounts of the main metabolite were found in pup urine, an effect of HCFC-123 or its metabolite on the pups via maternal milk is considered to be a possible cause for their decreased weight gain.

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design.

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.

Expression of cyclin D1 and p53 and its correlation with proliferative activity in the spectrum of esophageal carcinomas induced after duodenal content reflux and 2,6-dimethylnitrosomorpholine administration in rats.

Alterations in expression of the p53 and cyclin D1 genes have been implicated in the development of esophageal carcinomas in both humans and animal models. We hypothesize that altered expression of cyclin D1 and p53 may be involved in the sequential development of esophageal carcinomas with glandular differentiation induced by the carcinogen, 2,6-dimethylnitrosomorpholine (DMNM) in rats with duodenal content reflux esophagitis. In the present study Sprague-Dawley rats were given DMNM 15 days after performing an esophago-jejunostomy in order to induce chronic duodenal content reflux esophagitis. Expression and localization of p53, cyclin D1 and Ki-67 were examined by immunohistochemical analyses. Twenty of 24 animals developed different types of esophageal carcinomas, including pure squamous carcinoma, adenosquamous carcinoma and pure adenocarcinoma. Undifferentiated basaloid areas were frequently observed in these tumors. Cyclin D1 overexpression was observed in hyperplastic lesions and increased through dysplasia and in undifferentiated areas of infiltrating carcinoma. Cyclin D1 expression coincided with increased Ki-67 expression and decreased along with cell differentiation. The p53 immunohistochemical pattern was parallel to that of cyclin D1, although the percentage of positive cells was usually smaller in all lesions and increased p53 expression started at the dysplastic stage. These findings suggest that overexpression of cyclin D1 may be an early event in DMNM-induced rat esophageal tumorigenesis, causing increased proliferation of esophageal stem cells. Abnormal p53 expression may then be required to promote the development of neoplastic transformation from dysplastic epithelium through invasive phenotype, being more evident in cancer cells with squamous differentiation.

Inhalation tolerance study for p-aramid respirable fiber-shaped particulates (RFP) in rats.

This study was designed to assess the lung clearance function in rats after subchronic exposure to p-aramid respirable fiber-shaped particulates (RFP). Male Wistar rats were exposed 6 hrs/day, 5 days/week for 3 months to 50, 200, and 800 RFP/ml measured by scanning electron microscopy (SEM). Recovery effects were followed up through 9 months postexposure. The retention of RFP (length > 5 microm) was about 25 x 10(6) RFPs per lung in the low dose group after 3 months of exposure. The corresponding values in the medium and high dose groups amounted to overproportionally higher values of 122 x 10(6) and 576 x 10(6) RFPs per lung, respectively. A decrease in the length of the retained RFPs over the 9-month recovery period was observed, indicating a breakage of long fibrils. Alveolar clearance half-times measured by gamma tracers indicated a dust overloading of lungs for the high dose group at 0 and 3 months postexposure. Bronchoalveolar lavage parameters revealed that p-aramid RFPs induced pronounced inflammatory effects in the high and medium dose groups. Histopathologically, slight fibrotic and hyperplastic lesions were observed in the medium and high dose groups directly after the end of exposure. The findings at the 3-month postexposure interval resulted in a reduction of inflammatory changes in the medium and high dose groups compared to the sacrifices upon cessation of exposure. No histopathologic effects were detected in the low dose group. In the high dose group the maximum functionally tolerated dose was exceeded. The No Observed Adverse Effect Level (NOAEL) of RFP was 50 RFP/ml as measured by SEM.

Investigation of Chronic Toxic and Carcinogenic Effects of Gasoline Engine Exhausts Deriving from Fuel without and with Ferrocene Additive.

Chronic toxic and carcinogenic effects of gasoline engine exhaust inhalation were investigated in rats. The exhaust from the combustion of commercial fuel containing 30 ppm ferrocene additive was compared to exhaust from the same fuel without ferrocene. This study was part of a procedure to get a special authorization for the use of ferrocene as gasoline additive according to the German Gasoline Lead Act. To generate the exhausts, pairs of engines of the same type and age were operated on computer-controlled test benches in a combined urban-freeway driving cycle. The engines were equipped with three-way catalysts and lambda sensors. Rats inhaled the exhausts after dilution at ratios of about 1.20 and 1:40 for 18 h/day, 5 days/wk for 12 mo (chronic toxicity study) or for 24 mo followed by 6 mo of clean air (carcinogenicity study). The limiting factor for the exhaust concentration was the relative humidity of the exposure atmosphere. At defined intervals, body weight and food consumption, parameters of clinical chemistry, hematology, bronchoalveolar lavage (BAL), and mechanical lung function were measured, as well as lung clearance and particle retention in the lungs. In the high-dose groups and the controls the complete organ/tissue spectrum was investigated histopathologically, and in the low-dose groups the respiratory tract. Only slight exposure-related effects could be detected, like a loss in the background iron content of the cell pellet of the bronchoalveolar lavage fluid and cytoplasmic inclusions and goblet-cell hyperplasias in the nasal cavity. Between the clean-air controls and the exhaust-exposed groups, no exposure-related differences occurred in body weight development, mortality incidences, or any of the clinical investigations. Ninety-two to 94% of the animals developed age-related tumors, predominantly in the mammary glands, uterus, adrenals, thyroid, and pituitary. In the respiratory tract a total of five tumors was found: one in the controls and four in the low-dose groups. No physical, chemical, or toxicological differences between the exhausts from fuel without or with ferrocene were demonstrated.

Ito cell tumor: immunohistochemical investigations of a rare lesion in the liver of mice.

In 2 lifespan transgeneration experiments using a total of 4,682 CBA/J mice, we observed uncommon lipomatous lesions in the livers of 8 mice independent of the treatment. Macroscopically, the lesions were described as pale white areas (2) or nodules (6) during necropsy. The lesions ranged from 1 to 15 mm in diameter. Microscopically, the lesions consisted of nodular aggregations of round to spindle-shaped cells that partly caused distinct compression of the adjacent hepatic parenchyma. The tumor cells were smaller than hepatocytes and had dark oval nuclei. Many of the more spherical cells contained clear vacuoles of various sizes, which were shown to be lipid droplets by oil red O staining. In addition to Gomori's silver and Masson's trichrome staining, several immunohistochemical stains were used to characterize the origin of the proliferating cells. Tumor cells were labeled by vimentin, actin, desmin, and proliferating cell nuclear antigen. The 2 cell phenotypes showed similar staining characteristics. Increased amounts of laminin and tenascin, 2 extracellular matrix proteins of the liver, were detected within these neoplasms. Summarizing, we suggest that these tumors are of Ito cell origin.

Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice.

A lifetime experiment using 4279 CBA/J mice was carried out to investigate whether the pre-conceptual exposure of sperm cells to X-ray radiation or urethane would result in an increased cancer risk in the untreated progeny, and/or increased susceptibility to cancer following exposure to a promoting agent. The study consisted of four main groups, namely a control group (saline), a urethane group (1 mg/g body wt) and two X-ray radiation groups (1 Gy, 2 Gy). At 1, 3 and 9 weeks after treatment, the males of these four parental groups were mated with untreated virgin females. The offspring of each parental group was divided into two subgroups: one received s.c. urethane (0.1 mg/g body wt once) as a promoter, the other saline, at the age of 6 weeks. All animals were evaluated for the occurrence of tumours. K-ras oncogene and p53 tumour suppressor gene mutations were investigated in frozen lung tumour samples. The female offspring of male parents exposed to X-rays 1 week before their mating showed a trend towards a higher tumour incidence of the haematopoietic system than the F1 controls. In addition, a higher percentage of bronchioloalveolar adenocarcinomas in male offspring born to irradiated paternals mated 1 week after X-ray treatment points to a plausible increased sensitivity of post-meiotic germ cell stages towards transgenerational carcinogenic effects. On the other hand, no increased tumour incidence and malignancy were observed in the offspring born to irradiated paternals mated 3 and 9 weeks after X-ray treatment. Paternal urethane treatment 1, 3 and 9 weeks prior to conception did not result in significantly altered incidence or malignancy of tumours of the lung, liver and haematopoietic tissue in the offspring. K-ras mutations increased during tumour progression from bronchioloalveolar hyperplasia to adenoma. Codon 61 K-ras mutations were more frequent in lung tumours of urethane-promoted progeny from irradiated parents than from control parents. P53 mutations were absent from these lung alterations.

Quality control of three methods for lung tumorigenesis studies.

Many variables influence experimental results obtained from laboratory animal studies. One of the variables is tissue sampling for the detection of lesions. The contribution of different levels of sampling to the variability in reported tumour rates was evaluated in a tumorigenesis study using 1872 CBA/J mice. The number of lung neoplasms was estimated by three methods and the results compared. These methods were: 1. counting the macroscopically visible nodules, 2. microscopical examination of macroscopically-detected nodules and one histological section of each lung lobe, cut at the level of the bronchi (common method) and 3. microscopical examination as in method number 2 and additional microscopic examination of step-sections (200 microm interval) of the remaining lung tissue beginning at the level of the bronchi. Analysis using only macroscopic examination (method 1) showed that 40% (747/1872) of the animals had single or multiple nodules (i.e. tumour suspicious areas) in the lungs. When combined with microscopic examination (method 2), primary lung tumours were diagnosed in only 586 animals (31%). Evaluation by gross examination alone therefore gave an apparent overestimation of lung tumours compared to microscopic evaluation of grossly visible nodules. This was found to be due to a significant number of mice having nodules formed by processes other than primary lung tumours (i.e. non-specific inflammation, alveolar histiocytosis, focal hyperplasia of the alveolar epithelium, lymphoma infiltration or tumour metastases). On the other hand, in the more thorough sectioning of the lungs (method 3), primary lung tumours were detected in 712/1872 animals (38%). Additionally, these three different methods influenced the results with regard to the tumour multiplicity in each tumour-bearing animal.

Pulsed electrochemical detection of sulfur-containing antibiotics following high performance liquid chromatography.

Pulsed electrochemical detection (PED) following reversed-phase chromatography has been applied to the direct detection of sulfur-containing antibiotics, specifically, penicillins, cephalosporins, and lincomycin. The compounds are detected sensitively and selectively without the need for derivatization. Integrated pulsed amperometric detection (IPAD) yields limits of detection lower than UV detection for these compounds. Detection limits using an optimized IPAD waveform are typically 10 ppb or less. The high selectivity of PED for thiocompounds reduces sample preparation. This work is applied to the determination of penicillin and related analogues in various pharmaceutical formulations/preparations, including a chicken feed.

Assay for cephapirin and ampicillin in raw milk by high-performance liquid chromatography--integrated pulsed amperometric detection.

The FDA has issued guidelines governing the use of antibiotics in cattle and routinely tests for the presence of antibiotics in milk. Unfortunately, these compounds are often difficult to detect by direct methods because they often lack a chromophore or fluorophore. Integrated pulsed amperometric detection (IPAD) following reversed-phase liquid chromatography is well-suited for this analysis because it is selective, sensitive, and direct; i.e., derivatization is not required. This work involves the development of a simple, rapid assay for the determination of beta-lactam antibiotic residues in milk using HPLC-IPAD, specifically, ampicillin and cephapirin. Since the analyst studied here are detectable by UV detection, a comparison between IPAD and UV detection will be made. Sample preparation schemes that involve the extraction of antibiotics of interest from the milk matrix and subsequent cleanup are an important aspect of this project. These procedures will be discussed in detail. In addition, analytical figures of merit and IPAD wave form optimization will be addressed.

Ki-ras gene mutations and absence of p53 gene mutations in spontaneous and urethane-induced early lung lesions in CBA/J mice.

Ki-ras and p53 genes are involved in human lung carcinogenesis; however, the role of these genes in experimental lung tumors is not well known. In our study, the CBA/J mouse strain was used to investigate the presence of Ki-ras and p53 alterations in lung carcinogenesis of spontaneous tumors and tumors induced with high and low doses of urethane (ethyl carbamate). To study the presence of these alterations in the early stages of lung carcinogenesis and in very small lung tumors, restriction fragment length polymorphism and single-strand conformation polymorphism analyses were performed on polymerase chain reaction-amplified DNA from microdissected tumoral and normal lung samples. Ki-ras gene mutations in codons 12 and 61 were detected in all types of lung lesions, even in small and preneoplastic lesions, and their incidence increased with progression from lung hyperplasias (18%) to adenomas (75%) and to carcinomas (80%). Urethane exposure, in both high and low doses, increased the incidence of Ki-ras mutations in lung tumors, especially in adenomas. The presence of Ki-ras gene mutations in very small urethane-induced lung tumors and the absence of hyperplasias among the treated-group lesions may indicate that urethane accelerates tumoral progression. No p53 mutations were detected in exons 5-8 in any of the epithelium-derived lung tumors. Only one p53 mutation in exon 5 was found in a spontaneous lymphoma. Therefore, p53 mutations do not seem to cooperate with Ki-ras gene mutations or represent an alternative molecular pathway in murine carcinogenesis.

Subcutaneous soft tissue tumours at the site of implanted microchips in mice.

An experiment using 4279 CBA/J mice of two generations was carried out to investigate the influence of parental preconceptual exposure to X-ray radiation or to chemical carcinogens. Microchips were implanted subcutaneously in the dorsolateral back for unique identification of each animal. The animals were kept for lifespan under standard laboratory conditions. In 36 mice a circumscribed neoplasm occurred in the area of the implanted microchip. Females were significantly more frequently affected than male mice. An influence of age or different treatment on the s.c. tumour incidence in two mice generations could not be observed. Macroscopically, firm, pale white nodules up to 25 mm in diameter with the microchip in its center were found. Microscopically, soft tissue tumours such as fibrosarcoma and malignant fibrous histiocytoma were detected.

Cystic squamous cell carcinomas in the lungs of Syrian golden hamsters induced by coal oven flue exhaust mixed with pyrolized tar pitch in combination with benzo(a)pyrene.

Among a variety of induced pulmonary tumours, cystic squamous cell carcinomas were observed in five Syrian hamsters that inhaled a mixture of pyrolized tar pitch with coal oven flue exhaust (PCE) and additionally received intratracheal injections of benzo(a)pyrene. The histological appearance of these particular tumours is described, compared to similar tumour types in rats and the susceptibility of both species to inert particles is discussed.

Thiodiacetic acid--a metabolite of Ethylene oxide.

Urine samples of premature babies contain high amounts of thiodiacetic acid (TDA). Since these pre-term infants are exposed to an increased oxygen atmosphere in the incubator, we supposed that these high levels of thiodiacetic acid might be produced from ethylene, generated in the course of lipid peroxidation processes. Considering that conversion of ethylene to ethylene oxide (EO) is well known in biology we investigated whether ethylene oxide is metabolised to thiodiacetic acid or not. Therefore Sprague-Dawley rats and NMRI mice were exposed to ethylene oxide for six hours. Urine specimens were collected after exposure and the amount of thiodiacetic acid was determined by gas chromatography/mass spectrometry. The quantity of excreted TDA increased enormously compared to control samples. So thiodiacetic acid seems to be a metabolite of ethylene oxide in vivo.

Spontaneous myelofibrosis in castrated and ovariectomized NMRI mice.

The histopathological appearance of myelofibrosis of the bone marrow is described in aged castrated males, ovariectomized females and in male and female control NMRI mice. The highest incidence of the lesion was observed in ovariectomized and female control mice where more than 90% of the animals were affected. The presence of myelofibrosis in the bone marrow of ovariectomized females and castrated males indicates that estrogens may not play a major role in the development of the lesion and other hormonal disturbances must also be considered.

The carcinogenic potency of carbon particles with and without PAH after repeated intratracheal administration in the rat.

The role of carcinogenic PAH in soot- and carbon black-related lung tumour induction in rats was investigated after intratracheal administration of carbon blacks (CB) and two types of diesel soot (DS), either as original or as toluene extracted particles. The total particle dose per animal was 15 mg subdivided into 16-17 weekly applications. There was one vehicle control and two groups were treated with a total dose of either 30 or 15 mg pure BaP as positive control. The main tumour results were: (a) original DS induced a higher tumour rate than extracted DS; (b) the carcinogenic potency of extracted CB probably depends on the size of the primary carbon particles and on the specific surface area of the particles; (c) extracted DS covered with 11 micrograms BaP per mg carbon particles caused a lower lung tumour rate than original DS containing only 0.9 ng BaP per mg, but a variety of other PAH and NO2-PAH; (d) a total dose of 15 mg pure BaP caused a lung tumour rate very similar to that of 30 mg extracted DS, 15 mg original DS or 15 mg Printex 90T CB extracted or covered with approximately 29.5 micrograms BaP per mg CB.

Absence of effect of caffeine on the thyroid in the Syrian golden hamster: results of a 90-day study.

Caffeine in drinking water was offered ad lib. to male and female Syrian golden hamsters (Mesocricetus auratus W) for 90 days. Animals were randomly assigned to three dose groups (91.3, 274 and 822 mg/litre) and one control group (filtered tap water), each consisting of 20 male and 20 female animals. In relation to body weight, mean caffeine consumption was higher in females (low dose: 14.7; medium dose: 50.8; high dose: 104.8 mg/kg body weight/day) than males (low dose: 9.0; medium dose: 24.6; high dose: 65.2 mg/kg body weight/day). Caffeine in plasma was measured after 3 days, 3 wk and 3 months of treatment. As expected from calculation of the caffeine intake, mean values were higher in females (low dose: 0.6; medium dose: 3.6; high dose: 7.2 mg/litre) than in males (low dose: 0.4; medium dose: 0.7; high dose: 2.9 mg/litre). After 3 days of treatment, a transient, non-dose-related increase in mean (SEM) tri-iodothyronine (n=10) was found in the medium and high-dose (751+/-23 and 742+/-25 ng/litre, respectively) groups of males compared with that in the controls (610+/-39 ng/litre)(P<0.05). The values measured at later time points (days 24 and 91) were similar in all groups. No treatment-related changes were found in thyroxine (days 3, 24 and 91) and other clinicochemical analytes (day 91), absolute and relative adrenal weight (day 91), gross pathology and thyroid histopathology (day 91). In conclusion, no signs of thyroid toxicity of caffeine were observed in the Syrian golden hamster.