Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

A novel, integrated in vitro carcinogenicity test to identify genotoxic and non-genotoxic carcinogens using human lymphoblastoid cells.

Human exposure to carcinogens occurs via a plethora of environmental sources, with 70-90% of cancers caused by extrinsic factors. Aberrant phenotypes induced by such carcinogenic agents may provide universal biomarkers for cancer causation. Both current in vitro genotoxicity tests and the animal-testing paradigm in human cancer risk assessment fail to accurately represent and predict whether a chemical causes human carcinogenesis. The study aimed to establish whether the integrated analysis of multiple cellular endpoints related to the Hallmarks of Cancer could advance in vitro carcinogenicity assessment. Human lymphoblastoid cells (TK6, MCL-5) were treated for either 4 or 23 h with 8 known in vivo carcinogens, with doses up to 50% Relative Population Doubling (maximum 66.6 mM). The adverse effects of carcinogens on wide-ranging aspects of cellular health were quantified using several approaches; these included chromosome damage, cell signalling, cell morphology, cell-cycle dynamics and bioenergetic perturbations. Cell morphology and gene expression alterations proved particularly sensitive for environmental carcinogen identification. Composite scores for the carcinogens' adverse effects revealed that this approach could identify both DNA-reactive and non-DNA reactive carcinogens in vitro. The richer datasets generated proved that the holistic evaluation of integrated phenotypic alterations is valuable for effective in vitro risk assessment, while also supporting animal test replacement. Crucially, the study offers valuable insights into the mechanisms of human carcinogenesis resulting from exposure to chemicals that humans are likely to encounter in their environment. Such an understanding of cancer induction via environmental agents is essential for cancer prevention.

A comparison of the genotoxicity of benzo[a]pyrene in four cell lines with differing metabolic capacity.

Benzo[a]pyrene (B[a]P) is a known genotoxin and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on genotoxicity of B[a]P. Investigating and better understanding the biological significance of low level chemical exposures will help improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines (AHH-1, MCL-5, TK6 and HepG2) with differential expression of the CYP450 enzymes CYP1A1, 1B1 and1A2 involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 than AHH-1 which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and HPRT assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, linear increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5µM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed linear increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1 respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.

Functional activity but not gene expression of toll-like receptors is decreased in the preterm versus term human placenta.

INTRODUCTION: Toll-like receptor (TLR) activity within gestation-associated tissues might have a role in normal pregnancy progression as well as adverse obstetric outcomes such as preterm birth (PTB). METHODS: The expression and activity of TLRs 1-9 in placentas collected following preterm vaginal delivery after infection-associated preterm labour (IA-PTL) at 25-36 weeks of gestation (preterm-svd, n = 10) were compared with those obtained after normal vaginal delivery at term (term-laboured; n = 17). Placental explants were cultured in the presence of agonists for TLR2, 3, 4, 5, 7, 8 and 9 and cytokine production after 24 h examined. Expression of TLR transcripts was determined using real time quantitative PCR. RESULTS: Reactivity to all agonists except CpG oligonucleotides was observed indicating that other than TLR9 all of the receptors studied yielded functional responses both term and preterm. Significantly less TNFα and IL-6, but not IL-10, were produced by preterm than term samples in response to all TLR agonists. Changes in TLR mRNA expression did not underlie functional differences in the preterm and term groups; nor does a pre-exposure/tolerance model mimic this finding. While glucocorticoids suppressed cytokine production in an in vitro model using term tissue the association between lower gestational age and decreased cytokine outputs suggests a temporally regulated response. DISCUSSION: Pro-inflammatory cytokine output in response to multiple TLR ligands was decreased in the preterm compared to the term placenta but gene expression for each TLR tended to be similar. Reduced cytokine production by the preterm placenta in response to stimulation of TLRs therefore must be regulated at the post-transcriptional level in a gestational age dependent manner.

Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells.

Micronucleus (MN) induction is an established cytogenetic end point for evaluating structural and numerical chromosomal alterations in genotoxicity testing. A semi-automated scoring protocol for the assessment of MN preparations from human cell lines and a 3D skin cell model has been developed and validated. Following exposure to a range of test agents, slides were stained with 4'-6-diamidino-2-phenylindole (DAPI) and scanned by use of the MicroNuc module of metafer 4, after the development of a modified classifier for selecting MN in binucleate cells. A common difficulty observed with automated systems is an artefactual output of high false positives, in the case of the metafer system this is mainly due to the loss of cytoplasmic boundaries during slide preparation. Slide quality is paramount to obtain accurate results. We show here that to avoid elevated artefactual-positive MN outputs, diffuse cell density and low-intensity nuclear staining are critical. Comparisons between visual (Giemsa stained) and automated (DAPI stained) MN frequencies and dose-response curves were highly correlated (R (2) = 0.70 for hydrogen peroxide, R (2) = 0.98 for menadione, R (2) = 0.99 for mitomycin C, R (2) = 0.89 for potassium bromate and R (2) = 0.68 for quantum dots), indicating the system is adequate to produce biologically relevant and reliable results. Metafer offers many advantages over conventional scoring including increased output and statistical power, and reduced scoring subjectivity, labour and costs. Further, the metafer system is easily adaptable for use with a range of different cells, both suspension and adherent human cell lines. Awareness of the points raised here reduces the automatic positive errors flagged and drastically reduces slide scoring time, making metafer an ideal candidate for genotoxic biomonitoring and population studies and regulatory genotoxic testing.

Pro-oxidant induced DNA damage in human lymphoblastoid cells: homeostatic mechanisms of genotoxic tolerance.

Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H(2)O(2)), potassium bromate (KBrO(3)), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H(2)O(2) and KBrO(3), but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair-focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H(2)O(2)-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H(2)O(2) dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in "genotoxic tolerance."

Thymic stromal lymphopoietin is present in human breast milk.

Thymic stromal lymphopoietin (TSLP) has emerged as a potential key mediator of both the initiation and maintenance of Th2 responsiveness and thereby the allergic inflammatory cascade. Given the possible role of TSLP in initiating Th2-mediated allergic disease more detailed investigation of TSLP expression and responsiveness in early life is warranted. Therefore, the presence of TSLP in breast milk was evaluated. Defatted human breast milk collected at 3-5 days and 11-26 days postpartum (n=22/group) and commercially available ready-made baby formula (n=3) were analysed for TSLP with a specific ELISA. TSLP was detectable in breast milk but not in commercial ready-made formulas. Levels in breast milk were significantly higher (p<0.001) at 3-5 days than at 11-26 days postpartum. The potential role of breast milk TSLP in the development of immune function within the neonatal gastrointestinal tract and the development of allergy in childhood remain to be determined.

Expression and activity of Toll-like receptors 1-9 in the human term placenta and changes associated with labor at term.

Inflammatory processes are involved in the initiation and maintenance of labor, suggesting that Toll-like receptor (TLR) activity within gestation-associated tissues, such as the placenta, might contribute to the process of parturition. Expression of transcripts for TLR1-TLR10 was examined in term (>37 wk of gestation) human placentas collected in the absence of labor (elective caesarean sections; ECS; n = 11) and after the completion of labor (normal vaginal delivery; NVD; n = 12). Placental explants were cultured in the presence of agonists for TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9, and cytokine production after 24 h was examined. All placentas expressed transcripts for TLR1-TLR10. Reactivity to all agonists except CpG oligonucleotides was observed, indicating that, other than TLR9, all of the receptors studied yielded functional responses. Placental explants prepared from NVD placentas (n = 17) produced significantly more TNFA in response to lipopolysaccharide (TLR4 agonist) and resiquimod (TLR7/8 agonist) than explants from ECS placentas (n = 17). In contrast, gene expression analysis revealed that only transcripts for TLR2 and TLR5 were significantly elevated in association with labor. The human term placenta expresses a variety of functional TLRs, indicating that this family of receptors has an important role in parturition via as yet undetermined cell types and signaling pathways.