Validation of an air-liquid interface toxicological set-up using Cu, Pd, and Ag well-characterized nanostructured aggregates and spheres.

ABSTRACT: Systems for studying the toxicity of metal aggregates on the airways are normally not suited for evaluating the effects of individual particle characteristics. This study validates a set-up for toxicological studies of metal aggregates using an air-liquid interface approach. The set-up used a spark discharge generator capable of generating aerosol metal aggregate particles and sintered near spheres. The set-up also contained an exposure chamber, The Nano Aerosol Chamber for In Vitro Toxicity (NACIVT). The system facilitates online characterization capabilities of mass mobility, mass concentration, and number size distribution to determine the exposure. By dilution, the desired exposure level was controlled. Primary and cancerous airway cells were exposed to copper (Cu), palladium (Pd), and silver (Ag) aggregates, 50-150 nm in median diameter. The aggregates were composed of primary particles <10 nm in diameter. For Cu and Pd, an exposure of sintered aerosol particles was also produced. The doses of the particles were expressed as particle numbers, masses, and surface areas. For the Cu, Pd, and Ag aerosol particles, a range of mass surface concentrations on the air-liquid interface of 0.4-10.7, 0.9-46.6, and 0.1-1.4 µg/cm2, respectively, were achieved. Viability was measured by WST-1 assay, cytokines (Il-6, Il-8, TNF-a, MCP) by Luminex technology. Statistically significant effects and dose response on cytokine expression were observed for SAEC cells after exposure to Cu, Pd, or Ag particles. Also, a positive dose response was observed for SAEC viability after Cu exposure. For A549 cells, statistically significant effects on viability were observed after exposure to Cu and Pd particles. The set-up produced a stable flow of aerosol particles with an exposure and dose expressed in terms of number, mass, and surface area. Exposure-related effects on the airway cellular models could be asserted.

Cadmium and mercury exposure over time in Swedish children.

PURPOSE: Knowledge about changes in exposure to toxic metals over time remains very sparse, in particular for children, the most vulnerable group. Here, we assessed whether a reduction in environmental pollution with cadmium (Cd) and mercury (Hg) caused a change in exposure over time. In total, 1257 children (age 4-9) in two towns in Sweden were sampled once in 1986-2013. Blood concentrations of Cd (b-Cd; n=1120) and Hg (b-Hg; n=560) were determined. RESULTS: The median b-Cd was 0.10 (geometric mean 0.10; range 0.010-0.61) µg/L and b-Hg was 0.91 (geometric mean 0.83; range 0.021-8.2) µg/L. Children living close to a smelter had higher b-Cd and b-Hg than those in urban and rural areas. There was no sex difference in b-Cd or b-Hg, and b-Cd and b-Hg showed no significant accumulation by age. b-Cd decreased only slightly (0.7% per year, p<0.001) over the study period. In contrast, b-Hg did show a clear decrease over the study period (3% per year, p<0.001). CONCLUSIONS: The exposure to Cd was very low but still might increase the risk of disease later in life. Moreover, b-Cd only showed a minor decrease, indicating that Cd pollution should be further restricted. b-Hg was relatively low and decreasing, probably because of reduced use of dental amalgam and lower Hg intake from fish. The b-Cd and b-Hg levels decreased much less than the levels of lead in the blood as previously found in the same children.

Genome-wide gene expression profiling of low-dose, long-term exposure of human osteosarcoma cells to bisphenol A and its analogs bisphenols AF and S.

The bisphenols AF (BPAF) and S (BPS) are structural analogs of the endocrine disruptor bisphenol A (BPA), and are used in common products as a replacement for BPA. To elucidate genome-wide gene expression responses, estrogen-dependent osteosarcoma cells were cultured with 10 nM BPA, BPAF, or BPS, for 8 h and 3 months. Genome-wide gene expression was analyzed using the Illumina Expression BeadChip. Three months exposure had significant effects on gene expression, particularly for BPS, followed by BPAF and BPA, according to the number of differentially expressed genes (1980, 778, 60, respectively), the magnitude of changes in gene expression, and the number of enriched biological processes (800, 415, 33, respectively) and pathways (77, 52, 6, respectively). 'Embryonic skeletal system development' was the most enriched bone-related process, which was affected only by BPAF and BPS. Interestingly, all three bisphenols showed highest down-regulation of genes related to the cardiovascular system (e.g., NPPB, NPR3, TXNIP). BPA only and BPA/BPAF/BPS also affected genes related to the immune system and fetal development, respectively. For BPAF and BPS, the 'isoprenoid biosynthetic process' was enriched (up-regulated genes: HMGCS1, PDSS1, ACAT2, RCE1, DHDDS). Compared to BPA, BPAF and BPS had more effects on gene expression after long-term exposure. These findings stress the need for careful toxicological characterization of BPA analogs in the future.

Arsenic exposure in early pregnancy alters genome-wide DNA methylation in cord blood, particularly in boys.

Early-life inorganic arsenic exposure influences not only child health and development but also health in later life. The adverse effects of arsenic may be mediated by epigenetic mechanisms, as there are indications that arsenic causes altered DNA methylation of cancer-related genes. The objective was to assess effects of arsenic on genome-wide DNA methylation in newborns. We studied 127 mothers and cord blood of their infants. Arsenic exposure in early and late pregnancy was assessed by concentrations of arsenic metabolites in maternal urine, measured by high performance liquid chromatography-inductively coupled plasma mass spectrometry. Genome-wide 5-methylcytosine methylation in mononuclear cells from cord blood was analyzed by Infinium HumanMethylation450K BeadChip. Urinary arsenic in early gestation was associated with cord blood DNA methylation (Kolmogorov-Smirnov test, P-value<10-15), with more pronounced effects in boys than in girls. In boys, 372 (74%) of the 500 top CpG sites showed lower methylation with increasing arsenic exposure (r S -values>-0.62), but in girls only 207 (41%) showed inverse correlation (r S -values>-0.54). Three CpG sites in boys (cg15255455, cg13659051 and cg17646418), but none in girls, were significantly correlated with arsenic after adjustment for multiple comparisons. The associations between arsenic and DNA methylation were robust in multivariable-adjusted linear regression models. Much weaker associations were observed with arsenic exposure in late compared with early gestation. Pathway analysis showed overrepresentation of affected cancer-related genes in boys, but not in girls. In conclusion, early prenatal arsenic exposure appears to decrease DNA methylation in boys. Associations between early exposure and DNA methylation might reflect interference with de novo DNA methylation.

Long-term lead elimination from plasma and whole blood after poisoning.

OBJECTIVE: Blood lead (B-Pb), one of the most used toxicological biomarker all kind, has serious limitations. Thus, the objective is to evaluate whether plasma lead (P-Pb) is more adequate. METHODS: A long-term follow-up study of five cases of lead poisoning. P-Pb was analysed by inductively coupled plasma mass spectrometry. Kinetics after end of exposure was modelled. RESULTS: P-Pb at severe poisoning was about 20 µg/L; haematological effects at about 5 µg/L. Biological half-time of P-Pb was about 1 month; B-Pb decay was much slower. CONCLUSION: P-Pb is a valuable biomarker of exposure to and risk, particularly at high exposure.

Telomeric associations correlate with telomere length reduction and clonal chromosome aberrations in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is characterized cytogenetically by frequent telomeric associations (tas). To explore the mechanisms behind the formation of tas in GCTB and to investigate their karyotypic consequences, the frequencies of tas and clonal aberrations other than tas in 20 GCTBs were compared to telomere length and status, as assessed by quantitative PCR, fluorescence in situ hybridization (FISH), and expression levels of four genes involved in telomere maintenance. Based on the G-banding results, the tumors were divided into two groups, one with a high frequency of tas and one with a low frequency. Clonal aberrations were found to be restricted to the group with a high level of tas, and the same group showed a significantly larger reduction in telomere length in tumor cells compared to peripheral blood cells. Furthermore, 65 out of 66 tas analyzed by FISH were negative for telomeric sequences. The expression levels of TERT, TERF1, TERF2, and POT1 did not correlate with telomere length or the frequency of tas. Thus, the present findings provide strong support for the notion that decreased telomere length is a prerequisite for tas in GCTBs and that the clonal changes occurring in GCTBs are derived from tas.

Analysis of the distribution and frequency of trisomy 7 in vivo in synovia from patients with osteoarthritis and pigmented villonodular synovitis.

Osteoarthritis (OA) and pigmented villonodular synovitis (PVNS) are disorders associated with trisomy 7. The aim of the present study was to determine the frequency and distribution of the cells with +7 in vivo by analyzing sections of paraffin-embedded synovia from patients affected by OA, PVNS, other forms of synovitis [hemorragic synovitis (HS) and chronic synovitis (CS)], and from individuals without joint disease. Fluorescence in situ hybridization (FISH), using a centromeric probe for chromosome 7, showed that the mean frequency of trisomic nuclei in 5-microm sections was highest in PVNS (9.0%), followed by CS (5.9%), OA (5.6%), and HS (4.6%), whereas trisomic nuclei were rare (0.7%) in normal tissue. When 8-microm sections were studied, the frequencies of trisomic cells in OA and control synovia increased to 6.7% and 1.5%, respectively. Trisomic nuclei were found in all cases, including those for which cytogenetic analysis of short-term cultures had not disclosed any trisomic cells. Overall, the trisomic cells were scattered within the tissue. However, small clusters of cells with +7 were found in three cases. By hematoxylin-eosin staining of the slides used for FISH analysis it could be shown that the clustered trisomic cells were proliferating synoviocytes within villous extensions of the synovial membrane.

The tumor-associated gene HMGIC is expressed in normal and osteoarthritis-affected synovia.

Chromosomal rearrangements involving chromosome bands 12q13-15 are very frequent findings in benign solid tumors, and recently, the primary molecular target for these aberrations was identified as the gene HMGIC. However, mutations in this gene have also been observed in nonneoplastic tissues. In a previous study, we reported breakpoints within HMGIC of synovia affected by osteoarthritis (OA) in two cases with 12q15 aberrations. To analyze further the role of HMGIC in this disease, we have performed cytogenetic, fluorescent in situ hybridization (FISH), RNA, and protein expression analyses on synovial samples from patients with OA and individuals without signs of the disorder. Cytogenetic analysis of short-term cultured cells revealed clonal 12q13-15 aberrations in 2/36 cases of OA synovia and no rearrangement in any of the five controls. With FISH analysis, it was shown that the chromosomal breakpoints in the two aberrant cases were located outside the HMGIC locus. In contrast, at RNA and protein expression analyses, OA-affected as well as normal synovia displayed transcription and translation of the gene. We also analyzed whether immunoreactivity for HMGIC was associated with the proliferation-specific antigen Ki-67, but no correlation between the staining patterns of these proteins was observed. From the results of the present study, it is evident that expression of HMGIC cannot simply be considered a sign of neoplasia or an effect of proliferation.

Trisomy 7 accumulates with age in solid tumors and non-neoplastic synovia.

Trisomy 7 is a common finding in benign and malignant solid tumors, in several non-neoplastic lesions (for example, osteoarthritis and rheumatoid arthritis), and in apparently normal tissues as well, suggesting that the occurrence of +7 might be associated with factors other than the disease process itself. To find out whether the frequency of +7 varies with a patient's age, we cytogenetically analyzed short-term-cultured synovial samples from elderly persons without signs of arthritis and from young patients affected by juvenile chronic arthritis (JCA). In normal synovia, gain of a chromosome 7 was present as a clonal change in five of 10 cases and in single cells in four of the five remaining cases. In synovia from patients with JCA, cells with +7 were detected in only one of nine cases, representing the oldest patient in the series. Furthermore, we reviewed the cytogenetic literature on tumors of the brain, breast, colon, kidney, lung, skin, thyroid, and upper aerodigestive tract. In the majority (six of eight) of these tumor types, the frequency of cases displaying a clone with +7 as the sole aberration increased with age. Taken together, the results presented here suggest that the acquisition of trisomy 7 in some neoplastic and non-neoplastic tissues might be associated with age rather than with disease. The finding of a completely different frequency distribution in two of the tumor types (tumors of the brain and the thyroid gland), however, emphasizes the heterogeneity of +7 and indicates that other, possibly tissue-specific, factors might influence the occurrence of this mutation.

Cytogenetic polyclonality in hematologic malignancies.

The present study was undertaken to ascertain the frequency of cytogenetic polyclonality in various hematologic malignancies and to investigate whether morphologic subgroup, age, gender, or previous genotoxic exposure influences the incidence. Among 2,243 cytogenetically investigated hematologic malignancies, 10 acute myeloid leukemias (AML), 5 myelodysplastic syndromes (MDS), 2 acute lymphoblastic leukemias (ALL), 1 acute undifferentiated leukemia (AUL), 1 atypical Philadelphia chromosome-negative (Ph-) chronic myeloid leukemia (CML), 1 chronic myeloproliferative disorder (CMD), and 1 chronic lymphoproliferative disorder (CLD) with karyotypically unrelated clones were identified, constituting 2.6% of AML, 1.6% of MDS, 0.8% of ALL, 13% of AUL, 9.1% of Ph- CML, 1.5% of CMD, and 2.8% of CLD with chromosomal abnormalities. In contrast to the cytogenetic features, the X-inactivation pattern was monoclonal in the two informative female patients that could be investigated. Among 17,733 karyotypically aberrant published cases surveyed, significant frequency differences (P < 0.001) were discerned: 1.7% of 6,526 AML, 3.4% of 2,391 MDS, 0.4% of 1,920 Ph+ CML, 2.9% of 856 CMD, 0.9% of 4,226 ALL, and 5.8% of 1,814 CLD displayed unrelated clones. The incidence of cytogenetic polyclonality did not differ significantly among the MDS, CMD, or ALL subgroups, between males and females, between children (< 16 years) and adults, or between B- and T-cell ALL, whereas the frequencies varied among the AML FAB types (P < 0.05), among the different CLD entities (P < 0.001), and between B- and T-cell CLD (P < 0.001). Furthermore, the incidence was higher in therapy-related AML and MDS than in de novo AML and MDS (P < 0.001 and P < 0.01, respectively).

Rearrangement of the neoplasia-associated gene HMGIC in synovia from patients with osteoarthritis.

The occurrence of clonal chromosome aberrations in short-term cultures from synovia, osteophytes, and cartilage from patients with osteoarthritis (OA) was recently reported. Among these aberrations, a recurrent involvement of chromosome bands 12q13-15 in structural rearrangements was detected in both synovia and osteophytes. Chromosomal abnormalities of 12q13-15 are frequent among malignant and benign mesenchymal tumors, and it was recently demonstrated that the molecular target in these neoplasms is the HMGIC gene. In this study, we show by fluorescence in situ hybridization that HMGIC was disrupted by rearrangements of 12q15 in synovia from two patients with OA. The finding of HMGIC rearrangement in a lesion that is not traditionally regarded as neoplastic not only widens the spectrum of disorders that may be associated with altered function of this gene, but also provides further support for the notion that genetically rearranged cell populations are part of the OA process.

Polyclonal expansion of cells with trisomy 7 in synovia from patients with osteoarthritis.

Trisomy 7 as the single chromosome aberration has been found in a variety of neoplasms and in normal tissue in the proximity of tumors, as well as in non-neoplastic lesions. Recently, we described a nonrandom pattern of chromosome aberrations, in particular, a gain of chromosome 7, in synovia, cartilage, and osteophytes from patients with osteoarthritis. To study the clonal origin of trisomy 7 in osteoarthritis, multiple synovial samples were collected from five women, all of whom were informative heterozygotes with regard to the X-linked human androgen receptor gene (AR). From each case, three to four independent cell cultures were initiated. Trisomic cell populations were subcloned from the individual cultures, and it was established whether or not the same allele of AR was inactivated in trisomic cells from different parts of the same joint. The finding of a polyclonal X-inactivation pattern in two of the cases provides strong evidence that gain of an extra copy of chromosome 7 occurs independently in multiple cells.

Clonal chromosome aberrations are present in vivo in synovia and osteophytes from patients with osteoarthritis.

We have previously reported recurrent clonal chromosomal aberrations in synovia, osteophytes and articular cartilage from patients with osteoarthritis (OA). In particular, gain of chromosomes 5 and 7 was found to be strongly associated with OA. In order to exclude the possibility of in vitro artefacts, we studied three to four parallel, independent cultures from ten samples of synovia and three samples of osteophytes from ten women with primary OA. In all, 40 cultures were cytogenetically analysed, 39 of which had clonal chromosomal aberrations. The most common aberrations were +7 and +5 which were found in 38 and 12 cultures, respectively. There were striking karyotype similarities among the parallel cultures from each case. Out of a total of 83 clones, only 11 were unique for one culture, 7 from synovia and 4 from osteophytes. The genetic homogeneity among different cultures from the same patients excludes the possibility of in vitro artefacts and indicates a widespread distribution of the cytogenetically aberrant clones in vivo.

Slow deformation of intervertebral discs.

Intervertebral discs exhibit pronounced time-dependent deformations when subjected to load variations. These deformations are caused by fluid flow to and from the disc and by viscoelastic deformation of annulus fibres. The fluid flow is caused by differences between mechanical and osmotic pressure. A mechanical model of lumbar disc functions allows one to calculate both the extent of fluid flow and its implications for disc height as well as the role played by viscoelastic deformation of annulus fibres. From such calculations changes in body height are estimated. Experimental results already documented in the literature offer bases for the determination of the parameters involved. Body height variations are studied, both those related to normal diurnal rhythmicity and those related to somewhat exceptional circumstances. The normal diurnal fluid flow is found to be about +/- 40% of the disc fluid content late in the evening. Viscoelastic deformation of annulus fibres contributes approximately one quarter of the height change obtained after several hours normal activity, but dominates during the first hour.

On the mechanical behaviour of intervertebral discs.

The response to compression, shear, bending, and axial rotation of an intervertebral disc is studied by using a theoretical disc model. The annulus fibrosus is modelled by ten fibre layers with alternating fibre inclination. The nucleus pulposus as well as the substance filing the space between the fibre layers is assumed to consist of an incompressible fluid. The end-plates are assumed to bulge at action of the pressure in the fluid phase. Geometry of fibre layers and end-plates are chosen to agree as closely as possible with what is representative for lumbar discs. The results show considerable increase of the different stiffnesses at increasing levels of the axial load. They also show quite large fibre strains, well over 10% at motions within normal physiological limits.

Modeling of intervertebral discs.

A model with rotational symmetry of an intervertebral disc is studied. The nucleus pulposus is assumed to consist of an incompressible fluid, and the annulus fibrosus is modeled by 11 fiber layers with alternating fiber inclination. The spaces between the fiber layers are assumed to be filled with an incompressible fluid. The pressures in the fluid compartments, the fiber strains, and the bulge are calculated under different assumptions regarding fiber inclination, fiber force-elongation relation, initial fiber layer bulges, and so on. The agreement with experimental results is very good. Possible extensions of the model are briefly discussed.