Assessment of the genotoxicity of trichloroethylene in the in vivo micronucleus assay by inhalation exposure.

The in vivo genotoxic potential of trichloroethylene (TCE) was evaluated by examining the incidence of micronucleated polychromatic erythrocytes (MN-PCEs) in the bone marrow. Groups of male CD rats were exposed by inhalation to targeted concentrations of 0 (negative control), 50, 500, 2500 or 5000 ppm for 6 consecutive hours on a single day. The exposure concentrations were selected to overlap those employed by a published study that reported a 2- to 3-fold increase in the frequency of micronuclei in male rats following a single inhalation exposure to 5, 500 and 5000 ppm TCE for 6h but not following repeated exposure to similar concentrations. In addition, any treatment-related findings were assessed in the context of potential TCE-induced hypothermia. Clinical signs consistent with marked TCE-induced sedation were observed in rats exposed to 5000 ppm and subsequently three rats died prior to the end of the 6h exposure period. No remarkable changes in body temperature were observed in surviving animals monitored with transponders before and after exposures. There were no statistically significant increases in the frequencies of MN-PCEs in groups treated with the test material as compared to the negative controls. The positive control animals showed a significant increase in the frequency of MN-PCEs and a decrease in the relative proportion of PCEs among erythrocytes as compared to the negative control animals. There were no statistically significant differences in the per cent PCEs in groups treated with the test material. As no increase in the incidence of micronuclei was observed in any of the TCE exposure groups, kinetochore analyses were not performed. Under the experimental conditions used, TCE was considered to be negative in the rat bone marrow micronucleus test.

Does hydrogen peroxide exist "free" in biological systems?

Hydrogen peroxide (H2O2) can diffuse far from the site of production to intracellular locations where biological effects may be greater. The diffusion range is extended by H2O2 carriers formed spontaneously by hydrogen bonding with monomeric and polymeric compounds, including amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides. Hydrogen peroxide adducts (HPAs) are readily synthesized, e.g., crystalline histidine (His)-H2O2 adducts. An equilibrium exists between an adduct-forming compound and H2O2. The detection and relative stabilities of HPAs are measured by the degree of decomposition of H2O2 as influenced by test compounds in buffered solution competing with glucose or fructose for H2O2. The HPAs delay decomposition of H2O2 up to several hundredfold. The overall charge on an HPA, i.e., its ability to penetrate cell membranes, influences the cytotoxic and clastogenic effects of H2O2. Growth inhibition of Salmonella typhimurium LT2 by H2O2 is enhanced by neutral HPAs but decreased by anionic HPAs. Addition of catalase 1, 10, or 30 min after inoculation of S. typhimurium LT2 reduces or nearly eliminates partial growth inhibition by H2O2, but a neutral HPA, especially His-H2O2, transported H2O2 into the cells within 1 min, and in about 10 min completely inhibited growth. The stability of HPAs decreases with increasing pH or increasing temperature, while added Fe(II) in the presence and absence of EDTA accelerates H2O2 and HPA decomposition. Calculations indicate H2O2 hydrogen bonds with nucleic acid-base pairs with no apparent bond strain and energy stabilization comparable to normal hydrogen bonding.

Extreme population structuring in the threatened ghost bat, Macroderma gigas: evidence from mitochondrial DNA.

The ghost bat, Macroderma gigas, has undergone a major range contraction and is currently restricted around a few, highly disjunct maternity sites. The amount and distribution of mitochondrial DNA (mtDNA) variation within extant populations has been used to assess levels of current and historical maternal gene flow between these populations. An approximately 330 base pair fragment spanning a hypervariable area of the mtDNA control region was amplified and sequenced by using 22 individuals from four current ghost bat populations. The mean sequence diversity of 4.5% between populations was six times higher than that within populations (0.68%), and alleles within populations were monophyletic. Restriction enzyme analysis of amplified products from an additional 100 individuals revealed fixed allelic differences in the distribution of control region genotypes between the four populations. It is suggested that this extreme genetic subdivision is a consequence of long-term female philopatry. For the purposes of management each population should be treated as an independent entity. The depth of the genetic structuring suggests that the isolation of extant populations preceded the historical range contraction.

Reproductive performance links to fine-scale spatial patterns of female grey seal relatedness.

Fine-scale spatial patterns of female relatedness throughout the established grey seal breeding colony of North Rona, Scotland, were investigated by accurate mapping and spatially explicit analyses of a large sample (n = 262) of mothers using variation at nine microsatellite DNA loci. Local spatial autocorrelation analyses identified locations where seals were more highly related to the colony than average. These locations were also areas where the more successful females bred, were occupied first during each breeding season, were centrally placed locations of preferred habitat types and were likely to be the locations which were the first to be colonized historically. Mothers occupying such sites achieved higher than average pup growth rates, suggesting a founder fitness benefit.

The influence of parental relatedness on reproductive success.

The relationship between fitness and parental similarity has been dominated by studies of how inbreeding depression lowers fecundity in incestuous matings. A widespread implicit assumption is that adult fitness (reproduction) of individuals born to parents who are not unusually closely related is more or less equal. Examination of three long-lived vertebrates, the long-finned pilot whale, the grey seal and the wandering albatross reveals significant negative relationships between parental similarity and genetic estimates of reproductive success. This effect could, in principle, be driven by a small number of low quality, inbred individuals. However, when the data are partitioned into individuals with above average and below average parental similarity, we find no evidence that the slopes differ, suggesting that the effect is more or less similar across the full range of parental similarity values. Our results thus uncover a selective pressure that favours not only inbreeding avoidance, but also the selection of maximally dissimilar mates.

Cytotoxic and adaptive effects in rat nasal epithelium after 3-day and 13-week exposure to low concentrations of formaldehyde vapour.

To study in detail possible effects of low concentrations of formaldehyde on the nasal epithelium, Wistar rats were exposed to 0, 0.3, 1 and 3 ppm formaldehyde vapour for 6 h/day, 5 days/week during 3 days or 13 weeks, using in vivo [3H]thymidine labeling for cell proliferation studies, and light and electron microscopy for detecting morphological effects. Compound related histopathological nasal changes varying from epithelial disarrangement to epithelial hyperplasia and squamous metaplasia were found in the 3 ppm group, and were restricted to a small area of the anterior part of the nose which is normally covered with respiratory epithelium. These changes were confirmed by electron microscopy and were not observed in the other groups. Increased cell turnover in the same anterior location confirmed high mitotic activity in the 3 ppm group after 3 days and 13 weeks of exposure. At a slightly more posterior level in the nose a transient response in cell turnover was observed. After 3 days of exposure a nearly log-linear relationship was found between cell turnover and exposure concentration reaching a 10-fold increase in the 3 ppm group, and suggesting challenge of the mucociliary and/or regenerative defence systems not only at 3 ppm but also at 0.3 and 1 ppm. After 13 weeks of exposure mean turnover rates in all exposed groups were markedly lower than after 3 days, and the mean rates of the formaldehyde-exposed groups tended to be below that of the controls. The variation in turnover rate after 13 weeks had increased in a concentration related way, suggesting individual variation in adaptation. The most likely adaptive mechanism at this more posterior level of the nose seemed to be the mucociliary defence apparatus.

Subchronic (13-week) inhalation toxicity study of formaldehyde in male rats: 8-hour intermittent versus 8-hour continuous exposures.

Male Wistar rats were exposed for 13 weeks, 5 days a week to 0 (controls), 1 or 2 ppm formaldehyde continuously (8 h a day), or to 2 or 4 ppm formaldehyde interruptedly (eight 30-min exposure periods separated by 30-min non-exposure periods a day). Histopathological changes were only found in the nose of animals (interruptedly) exposed to 4 ppm formaldehyde and comprised an increased degree and incidence of disarrangement and squamous metaplasia accompanied by basal cell hyperplasia and occasionally by keratinization of the respiratory epithelium. Two ppm formaldehyde was the non-toxic effect level. Cell proliferation studies demonstrated a slightly higher cell turnover of the nasal respiratory epithelium exposed (interruptedly) to 4 ppm formaldehyde than in controls. It was concluded that under the conditions of repeated exposure to marginally cytotoxic concentrations during a period of 13 weeks the exposure concentration rather than the total 'dose' (= concentration x exposure time) determined the severity of the cytotoxic effects of formaldehyde on the nasal epithelium.

Influence of retinoids on the mutagenicity of cigarette-smoke condensate in Salmonella typhimurium TA98.

The effects of 3 different retinoids (all-trans-retinol, all-trans-retinoic acid, and all-trans-retinyl acetate) on the mutagenic activity of cigarette-smoke condensate were investigated in Salmonella typhimurium TA98. Neither an enhancing nor an inhibitory effect of the retinoids on the mutagnicity of cigarette-smoke condensate was observed.

Effect of cigarette-smoke condensate and norharman on the induction of SCEs by direct and indirect mutagens in CHO cells.

Cigarette-smoke condensate and norharman were investigated either alone or in combination with a number of direct or indirect mutagens for the induction of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Cigarette-smoke condensate and norharman induced SCEs in these cells, only in the presence of a metabolic activation system. The number of SCEs induced by the direct-acting mutagens mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine was decreased in the presence of cigarette-smoke condensate or norharman. However, cigarette-smoke condensate and norharman showed synergistic effects in combination with the indirect mutagens 2-acetylaminofluorene, 2-aminofluorene, N-hydroxy-acetylaminofluorene, 2-aminoanthracene and benzo[a]pyrene. No synergism was observed when CHO cells were treated simultaneously with cigarette-smoke condensate or norharman and the indirect mutagen cyclophosphamide.

Induction of sister-chromatid exchanges and chromosome aberrations by gamma-irradiated nucleic acid constituents in CHO cells.

Chinese hamster ovary (CHO) cells were exposed to different concentrations of hydrogen peroxide and to gamma-irradiated, oxygenated solutions of thymine, thymidine and 2-deoxy-D-ribose. By using a modified BrdUrd-labelling method sister-chromatid exchanges (SCEs) and chromosomal aberrations were scored in the same cell populations, one cycle after treatment. Irradiated, oxygenated solutions of 2-deoxy-D-ribose clearly induced SCEs and chromosomal aberrations. In comparison, hydrogen peroxide and irradiated solutions of thymine and thymidine were less effective in CHO cells.

Clastogenicity of isoamylene oxide to rat lymphocytes in culture.

The mutagenic activity of the aliphatic epoxide isoamylene oxide (2-methyl-2,3-epoxybutane) is not readily detectable in the standard Ames test. In this study, the clastogenic potential of isoamylene oxide was evaluated using an in vitro mammalian cell culture system. Approximately 48 h after establishing primary cultures of rat lymphocyte cultures, the cells were treated for 4 h with various concentrations of isoamylene oxide (50, 166.7, 500,, 1666.7, and 5000 micrograms/ml in the initial assay and 500, 1000, 2000, 3000, 4000, and 5000 micrograms/ml in the confirmatory assay). The cultures were harvested 24 h after termination of the treatment. Based upon the mitotic indices, cultures treated with the three highest concentrations in both the initial and confirmatory assays were evaluated to estimate the chromosomal aberration frequencies. Isoamylene oxide demonstrated a strong clastogenic activity in this assay: up to 29% aberrant cells (without gaps) were observed at the highest concentration analyzed. The presence of an external metabolic activation system (S9) did not seem to influence the magnitude of the response at the dose levels analyzed.

Monitoring the exposure of rats to 2-acetylaminofluorene by the estimation of mutagenic activity in excreta, sister-chromatid exchanges in peripheral blood cells and DNA adducts in peripheral blood, liver and spleen.

The sensitivity of various methods suitable for biomonitoring the exposure to genotoxicants was compared in an animal model. The results were related to the presence of genotoxic effects in the target organ. Groups of male Wistar rats were given one oral dose of 0, 0.1, 10 or 200 mg 2-acetylaminofluorene (2-AAF)/5 ml dimethyl sulphoxide/kg body weight. Peripheral blood cells, excreta, liver and spleen were collected at different time intervals after dosing. Mutagenicity in urine and extracts of faeces was determined using the Ames test with Salmonella typhimurium TA98 with and without S9 and with and without beta-glucuronidase. Genotoxic effects were studied by measuring DNA-adduct formation in lymphocytes, liver and spleen, and sister-chromatid exchanges (SCEs) in lymphocytes. DNA adducts were measured with immunochemical techniques and postlabelling methods. Mutagenicity in urine and faeces, collected during the first 24 h after treatment, was detected at 2-AAF doses of 1 mg/kg b.w. and higher. At these doses DNA adducts also became apparent in the liver, the main target organ for tumour induction by 2-AAF. The adduct detected appeared to be the N-(deoxyguanosin-8-yl)-2-AAF adduct. There was no evidence of the presence of any other types of DNA adducts. At doses of 1 and 10 mg/kg b.w. no mutagenicity was detected in excreta collected during the second and third day after dosing. The DNA-adduct level in liver cells of the 1 mg/kg b.w. group was maximal 24 h after dosing. At 200 mg/kg b.w. a delay in excretion of mutagenicity with urine and faeces was seen and at 10 and 200 mg/kg b.w. the amount of DNA adducts continued to increase with time after dosing. At 24 and 48 h after treatment with 10 mg, the adduct levels were of the same order of magnitude as those found after the 20-fold higher dose. This points to overloading of the metabolizing system which in combination with the enterohepatic circulation, may lead to an increased retention of 2-AAF in the body. A slightly increased incidence of SCEs of doubtful significance was seen in lymphocytes, but only at the very high dose of 200 mg/kg b.w. No DNA adducts could be detected in blood lymphocytes or spleen cells at any of the dose levels applied, either with the immunochemical or with the postlabelling method.(ABSTRACT TRUNCATED AT 400 WORDS)

Single-dose and 13-week repeated-dose neurotoxicity screening studies of chlorpyrifos insecticide.

Chlorpyrifos (CPF), a widely used organophosphate insecticide, was screened for neurotoxic effects in Fischer 344 rats using United States Environmental Protection Agency 1991 guidelines for single-dose and 13-wk repeated dose studies. The studies emphasized a functional observational battery (which included grip performance and hindlimb splay tests), automated motor activity testing and comprehensive neurohistopathology of perfused tissues. Doses of up to 100 mg/kg body weight in corn oil by gavage in the single-dose study and up to 15 mg/kg body weight/day in diet for 13 wk in the repeated dose study were administered. It is known that CPF and other phosphorothionates can be activated to the oxon in local (extrahepatic) tissues. Local activation could possibly cause different effects in different tissues with cholinergic innervation, and thereby create syndromes unique to each phosphorothionate according to their structure. Consequently, the conduct of CPF neurotoxicity screening studies by contemporary guidelines offered opportunity to characterize the CPF over-exposure syndrome in rats. Single-dose high levels of oral exposure to CPF caused a range of clinical signs characteristic of cholinergic overstimulation. Although there was no clinical evidence of wide differences in sensitivity of one cholinergic response versus another, motor dysfunction (incoordination etc.) was more prominent than other signs, for example soiling. Effects were much more apparent in females and regressed over several days. Effects were minimal in the 13-wk study, and there was no evidence of accumulation of toxicity during the 13 wk of daily dietary exposure. Motor activity was decreased at the high dose in males and females at wk 4, but was not significantly different from controls in subsequent weeks. The 'normalization' of motor activity later in the study was interpreted as tolerance to repeated administration of CPF. Comprehensive neuropathological examination revealed no treatment-related lesions in either study.

Seeing in the dark: molecular approaches to the study of bat populations.

Whilst the use of molecular genetic techniques is widespread in the fields of population and evolutionary biology, their application within the mammalian order Chiroptera neither reflects the species richness nor the ecological and behavioural diversity of the order. This is despite the fact that the Chiroptera are problematic to study using more direct observational techniques. Here, we standardize and synthesise the current data, assess the contribution of molecular research to the study of bat species and highlight the importance of its continued and expanded use. At an inter-population level, molecular studies have demonstrated a great diversity of population genetic structure within the order. Among populations of migratory species, genetic structure appears universally low, and hence seasonal movement is likely to be the prevailing influence. However, for sedentary species an array of factors including dispersal ability, extrinsic barriers to gene flow and historical events may determine the extent of genetic partitioning among populations. Intrinsic factors such as wing morphology or roost requirements may also influence population genetic structure in sedentary bat species, a proposal which requires further research. Molecular studies have also made important contributions towards an understanding of social organisation in bats. Evidence indicates that in many polygynous species male mating success does not translate directly into reproductive success, perhaps as a result of multiple mating by females. Estimates of relatedness within and genetic structure among colonies are, in general, very low; a finding which has important implications regarding theories concerning the formation and persistence of bat social groups. Molecular studies have provided new and important insights into the ecology of bats, and have opened up exciting and previously unexplored avenues of research. The data from these studies suggest not only a predictive framework for future studies, but also the use of genetic data in the management and conservation of bat species.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. I. A cell proliferation study.

The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a dose-dependent increase in labeling index (LI) during 12 days of culture. The basal cells were more sensitive to CSC exposure than non-basal cells during the first 6 to 8 culture days. However, in squamous metaplastic foci developing after culture day 6, both basal and non-basal cells in the mid-part of the epithelium were labeled. Physiological concentrations of all-trans retinol stimulated the non-basal LI and inhibited the basal cell LI. Compared with dimethylsulfoxide (DMSO), all retinol concentrations used in the present study inhibited the basal cell LI at each time point examined (4-12 days culture). Exposure of tracheal rings to retinol, either before or after exposure to CSC, or simultaneous exposure to retinol and CSC, clearly decreased the CSC-induced basal cell proliferative activity depending on the retinol concentration used. It is concluded from the present study that squamous metaplasia induced by vitamin A-deficiency or by CSC originates mainly from basal cells and that for the maintenance of these lesions, both basal and non-basal cells play a role. Furthermore, all-trans retinol inhibited CSC-induced basal cell proliferation.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. II. A histomorphological study.

The effects of all-trans retinol and cigarette smoke condensate (CSC) on tissue morphology and cellular differentiation were investigated in vitamin A-deprived tracheal epithelium cultured in vitamin A-and serum-free hormone-supplemented medium. Physiological retinol concentrations prevented the development of hyperplasia and squamous metaplasia with or without keratinization, and induced differentiation to mucous cells. Squamous metaplastic foci with keratinization were observed during 12 days of culture with low retinol concentrations and with dimethylsulfoxide (DMSO) which was accompanied by an increased number of basal and indeterminate cells. CSC induced a dose-related hyperplasia and irregularly shaped foci of squamous metaplasia with atypical epithelial proliferation. In non-metaplastic epithelium, CSC exposure increased the number of ciliated cells. Hyperplasia and squamous metaplasia were inhibited if the tracheal rings were first treated with retinol followed by CSC exposure, or if the tracheas were simultaneously treated with retinol and CSC. CSC-exposure prior to retinol treatment induced similar histomorphological alterations as CSC alone.

Localization of DNA adducts formed in the nasal cavity of the rat by the tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK).

The tissue localization of the DNA adducts O6- and 7-methylguanine induced in the nasal cavity by the nicotine-derived carcinogen 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK, 30 mg/kg intraperitoneally) has been investigated immunocytochemically in male Sprague-Dawley rats. Adduct-specific nuclear staining, indicative of the metabolic activation of NNK to a methylating compound, was observed in both respiratory and olfactory mucosa. In the respiratory epithelium, strong staining was generally observed in areas devoid of goblet cells. Less intense staining was observed both in the serous gland cells and their efferent ducts in the respiratory submucosa, whereas the mucous gland cells were unstained. In the olfactory mucosa, the sustentacular and basal cells of the olfactory epithelium were moderately stained; staining varied substantially from site to site. No DNA adduct was detected in the olfactory cells. Strong nuclear staining, similar to that in the respiratory mucosa, was observed in the cells of the Bowman glands of the olfactory submucosa. A similar distribution of methylated DNA bases in nasal tissues has been observed in rats after exposure to other N-nitrosamines and in Syrian hamsters after exposure to NNK. This finding may indicate that in man the same cell types undergo DNA adduct formation after exposure to NNK and other N-nitrosamines.

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide.

The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-3H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 micron, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-3H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration.