Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis.

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.

Determination using synchrotron radiation-based Fourier transform infrared microspectroscopy of putative stem cells in human adenocarcinoma of the intestine: corresponding benign tissue as a template.

The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 µm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as "distinct base region" spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations could point to a cancer SC phenotype; an approach capable of identifying their in situ location has enormous therapeutic applications.

Bimodal responses of cells to trace elements: insights into their mechanism of action using a biospectroscopy approach.

Understanding how organisms respond to trace elements is important because some are essential for normal bodily homeostasis, but can additionally be toxic at high concentrations. The inflection point for many of these elements is unknown and requires sensitive techniques capable of detecting subtle cellular changes as well as cytotoxic alterations. In this study, we treated human cells with arsenic (As), copper or selenium (Se) in a dose-response manner and used attenuated total reflection Fourier-transform infrared (ATR-FTIR) microspectroscopy combined with computational analysis to examine cellular alterations. Cell cultures were treated with As(V), Cu(2+) or Se(IV) at concentrations ranging from 0.001 mg L(-1) to 1000 mg L(-1) and their effects were spectrochemically determined. Results show that As(V) and Cu(2+) induce bimodal dose-response effects on cells; this is in line with hormesis-driven responses. Lipids and proteins seem to be the main cell targets for all the elements tested; however, each compound produced a unique fingerprint of effect. Spectral biomarkers indicate that all test agents generate reactive oxygen species (ROS), which could either stimulate repair mechanisms or induce damage in cells.

Evaluation of ATR-FTIR spectroscopy with multivariate analysis to study the binding mechanisms of ZnO nanoparticles or Zn2+ to Chelex-100 or metsorb.

Advancements in nanotechnology and the expected increases in production of commercial products with incorporated manufactured nanomaterials will very likely lead to increasing contamination of nanoparticles (NPs) in the environment. Though studying adverse impacts of NPs in the environment and their ecotoxicological fate and behavior is not new, limited information is available. A major challenge in this respect is the lack of a proper sampling technique that could provide data on concentrations of these materials in the environment. Diffusive gradient in thin-films (DGT) is a well-established method that can measure available concentrations of trace metals in soils and waters. Using this approach, different binding resins are employed as a sink to collect targeted chemicals during fixed times. Here, we examine the suitability of two common types of the DGT binding agents, commercially available Chelex-100 and Metsorb, to investigate whether these materials could irreversibly retain a model nanoparticle, ZnO, and if so, what would be the difference between bound ZnO NP and Zn(2+) ion. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was used to study the binding materials before and after exposure to ZnO NP and Zn(2+). Based on computational analysis using principal component analysis and linear discriminant analysis (PCA-LDA), it was demonstrated that both Chelex-100 and Metsorb form chemical bonds with ZnO NP and Zn(2+), however the binding mechanisms of these zinc species as inferred from their infrared (IR) spectra are statistically different (95% confidence level). The experimental results suggest that the binding resins hold ZnO NP with fewer and weaker chemical bonds compared to Zn(2+). This research shows the potential of the DGT method to measure available concentrations of nanoparticles in the environment and demonstrate how ATR-FTIR spectroscopy, when used with computational analysis, can differentiate between diverse chemical species that are simultaneously retained by the binding layer in a DGT device.

Alterations in the infrared spectral signature of avian feathers reflect potential chemical exposure: a pilot study comparing two sites in Pakistan.

Chemical contamination of ecosystems is a global issue with evidence that pollutants impact on living organisms in a harmful fashion. Developing sensor approaches that would allow the derivation of biomarkers or signatures of effect in target sentinel organisms and monitor environmental chemical contamination in a high throughput manner is of utmost importance. As biomolecules absorb infrared (IR), signature vibrational spectra related to structure and function can be derived. In light of this, we tested the notion that IR spectra of bird feathers might reflect environmental chemical contaminant exposure patterns. Feathers were collected from monospecific heronries of cattle egret based in two independent locations (Trimu vs. Mailsi) in the Punjab province of Pakistan; these sites were found to differ in their chemical contamination patterns. Feather samples were chemically analyzed for polychlorinated biphenyls, polybrominated diphenyl ethers, organochlorines and heavy metals. Attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy was employed to derive a spectral signature of individual feathers. Resultant IR spectra were then subjected to canonical correspondence analysis (CAA) to determine whether feather spectral signatures correlate to chemical exposure. Additionally, we explored if principal component analysis (PCA) and linear discriminant analysis (LDA) could be applied to distinguish site-specific differences; linear discriminant function (LDF) was also applied to classify sites. The sampled feathers varied in their chemical exposure patterns depending on whether they were sourced from one site associated with heavy metal exposure or the other which suggested high organic pollutant exposures. CCA of chemical and spectral data showed a correlation between spectral signatures and chemical exposure. PCA-LDA readily distinguished feathers from the two different sites. Discriminating alterations were identified and these were associated with protein and lipid regions in IR spectra. Additionally, LDF showed that the classification rate of spectral categories correlated well with the two chemical exposure patterns (93.6% for Trimu feathers and 91.77% for Mailsi feathers). This pilot study suggests that IR spectra derived from feathers reflect background chemical exposure and points to a novel monitoring tool for contamination.

Identification of benzo[a]pyrene-induced cell cycle-associated alterations in MCF-7 cells using infrared spectroscopy with computational analysis.

Chemical contaminants, such as benzo[a]pyrene (B[a]P), may modulate transcriptional responses in cells via the activation of aryl hydrocarbon receptor (AhR) or through responses to DNA damage following adduct formation. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be employed in a non-destructive fashion to interrogate the biochemical signature of cells via generation of infrared (IR) spectra. By applying to generated spectral datasets subsequent computational approaches such as principal component analysis plus linear discriminant analysis (PCA-LDA), derived data reduction is achieved to facilitate the visualization of wavenumber-related alterations in target cells. Discriminating spectral variables might be associated with lipid or glycogen content, conformational protein changes and phosphorylation, and structural alterations in DNA/RNA. Using this approach, we investigated the dose-related effects of B[a]P in MCF-7 cells concentrated in S- or G0/G1-phase. Our findings identified that in PCA-LDA scores plots a clear segregation of IR spectra was evident, with the major spectral alterations associated with DNA/RNA, secondary protein structure and lipid. Dose-related effects were observed and even with exposures as low as 10⁻9 M B[a]P, significant (P ≤ 0.001) separation of B[a]P-treated vs. vehicle control cells was noted. ATR-FTIR spectroscopy with computational analysis is a novel approach to identify the effects of environmental contaminants in target cells.

Concentration-dependent effects of carbon nanoparticles in gram-negative bacteria determined by infrared spectroscopy with multivariate analysis.

With increasing production of carbon nanoparticles (CNPs), environmental release of these entities becomes an ever-greater inevitability. However, many questions remain regarding their impact on soil microorganisms. This study examined the effects of long or short multiwalled carbon nanotubes (MWCNTs), C60 fullerene and fullerene soot in Gram-negative bacteria. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was applied to derive signature spectral fingerprints of effects. A concentration-dependent response in spectral alterations was observed for each nanoparticle type. Long or short MWCNTs and fullerene soot gave rise to similar alterations to lipids, Amide II and DNA. The extent of alteration varies with nanoparticle size, with smaller short MWCNTs resulting in greater toxicity than long MWCNTs. Fullerene soot was the least toxic. C60 results in the most distinct and largest overall alterations, notably in extensive protein alteration. This work demonstrates a novel approach for assaying and discriminating the effects of CNPs in target systems.

Diagnostic segregation of human brain tumours using Fourier-transform infrared and/or Raman spectroscopy coupled with discriminant analysis.

The most common initial treatment received by patients with a brain tumour is surgical removal of the growth. Precise histopathological diagnosis of brain tumours is to some extent subjective. Furthermore, currently available diagnostic imaging techniques to delineate the excision border during cytoreductive surgery lack the required spatial precision to aid surgeons. We set out to determine whether infrared (IR) and/or Raman spectroscopy combined with multivariate analysis could be applied to discriminate between normal brain tissue and different tumour types (meningioma, glioma and brain metastasis) based on the unique spectral "fingerprints" of their biochemical composition. Formalin-fixed paraffin-embedded tissue blocks of normal brain and different brain tumours were de-waxed, mounted on low-E slides and desiccated before being analyzed using attenuated total reflection Fourier-transform IR (ATR-FTIR) and Raman spectroscopy. ATR-FTIR spectroscopy showed a clear segregation between normal and different tumour subtypes. Discrimination of tumour classes was also apparent with Raman spectroscopy. Further analysis of spectral data revealed changes in brain biochemical structure associated with different tumours. Decreased tentatively-assigned lipid-to-protein ratio was associated with increased tumour progression. Alteration in cholesterol esters-to-phenylalanine ratio was evident in grade IV glioma and metastatic tumours. The current study indicates that IR and/or Raman spectroscopy have the potential to provide a novel diagnostic approach in the accurate diagnosis of brain tumours and have potential for application in intra-operative diagnosis.

Differential effects in mammalian cells induced by chemical mixtures in environmental biota as profiled using infrared spectroscopy.

Environmental contaminants accumulate in many organisms and induce a number of adverse effects. As contaminants mostly occur in the environment as mixtures, it remains to be fully understood which chemical interactions induce the most important toxic responses. In this study, we set out to determine the effects of chemical contaminants extracted from Northern Gannet (Morus bassanus) eggs (collected from the UK coast from three sampling years (1987, 1990, and 1992) on cell cultures using infrared (IR) spectroscopy with computational data handling approaches. Gannet extracts were chemically analyzed for different contaminants, and MCF-7 cell lines were treated for 24 h in a dose-related manner with individual-year extracts varying in their polybrominated diphenyl ether (PBDE) to polychlorinated biphenyl (PCB) ratios. Treated cellular material was then fixed and interrogated using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy; resultant IR spectra were computationally analyzed to derive dose-response relationships and to identify biomarkers associated with each contaminant mixture treatment. The results show distinct biomarkers of effect are related to each contamination scenario, with an inverse relationship with dose observed. This study suggests that specific contaminant mixtures induce cellular alterations in the DNA/RNA spectral region that are most pronounced at low doses. It also suggests alterations in the "biochemical-cell fingerprint" of IR spectra can be indicative of mixture exposures.

High contrast images of uterine tissue derived using Raman microspectroscopy with the empty modelling approach of multivariate curve resolution-alternating least squares.

Approaches that allow one to rapidly understand tissue structure and functionality in situ remain to be developed. Such techniques are required in many instances, including where there is a need to remove with a high degree of confidence positive tumour margins during surgical excision. As biological tissue has little contrast, gold standard confirmation of surgical margins is conventionally undertaken by histopathological diagnosis of tissue architecture via optical microscopy. Vibrational spectroscopy techniques, when coupled to sophisticated computational analyses, are capable of constructing bio-molecular contrast images of unstained tissue. To assess the relative applicability of a range of candidate algorithms to distinguish the in situ bio-molecular structures of a complex tissue, the empty modelling approach of multivariate curve resolution-alternating least squares (MCR-ALS) was compared to hierarchical cluster analysis (HCA) or principal component analysis (PCA). Such chemometric analyses were applied to Raman images of benign (tumour-adjacent) endometrium, stage I and stage II endometrioid cancer. Re-constructed images from the in situ bio-molecular tissue architectures highlighted features associated with glandular epithelium, stroma, glandular lumen and myometrium. Of the tested chemometric analyses, MCR-ALS provided the best bio-molecular contrast images, superior to those derived following HCA or PCA, with clear and defined margins of histological features. Iteratively-resolved spectra identified wavenumbers responsible for the contrast image. Wavenumbers 1234 cm(-1) (Amide III), 1390 cm(-1) (CH(3) bend), 1675 cm(-1) (Amide I/lipid), 1275 cm(-1) (Amide III), 918 cm(-1) (proline) and 936 cm(-1) (proline, valine and proteins) were responsible for generating the majority of the contrast within MCR-ALS-generated images. Applications of sophisticated computational analyses coupled with vibrational spectroscopy techniques have the potential to lend novel functionality insights into bio-molecular structures in vivo.

Derivation by infrared spectroscopy with multivariate analysis of bimodal contaminant-induced dose-response effects in MCF-7 cells.

Toxic responses to contaminants following exposure concentrations typically used in laboratory tests may not reflect how biological systems respond to lower environmental levels from which hormetic effect mechanisms have been suggested. We investigated the pattern of dose-response in mammalian cells to various environmental contaminants using a range of concentrations that span those that are environmentally relevant (10(-12)M to 10(-3)M). MCF-7 cell cultures were treated for 24 h with benzo[a]pyrene (B[a]P), lindane (γ-hexachlorocyclohexane), or polybrominated diphenyl ethers (PBDEs) congeners (47, 153, 183, and 209), then fixed in ethanol and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. Mode of action was further studied by examining if test agents stimulated cell growth or altered CYP1A1 expression. Bimodal dose response curves were observed when MCF-7 cells were treated with PBDEs or lindane. The first peak distribution was associated with lower doses (10(-12)M to 10(-9)M), while the second occurred only after MCF-7 cells were exposed to concentrations >10(-9)M. Cellular alterations associated with low-dose PBDEs were mainly due to lipid and secondary protein structural changes, whereas lindane induced DNA/RNA effects as well. In contrast, DNA-reactive B[a]P gave rise to a monotonic linear dose-response relationship and induced mainly DNA/RNA cellular changes. This study shows that environmentally realistic exposures to chemical contaminants can induce nonmonotonic dose-responses in cellular systems.

Sublethal genotoxicity and cell alterations by organophosphorus pesticides in MCF-7 cells: implications for environmentally relevant concentrations.

Organophosphorus pesticide (OPP) toxicity is believed to be mediated through inhibition of acetylcholinesterase (AChE). Given their widespread distribution in aquatic systems and their ability to undergo chemical transformation, their environmental impacts at sublethal concentrations in nontarget organisms have become an important question. We conducted a number of mammalian-cell genotoxic and gene expression assays and examined cellular biochemical changes that followed low-dose exposure of MCF-7 cells to fenitrothion, diazinon, and the aqueous degradate of diazinon, 2-isopropyl-6-methyl-4-pyrimidinol (IMP). After exposure to the OPPs at low concentrations (10(-12) M to 10(-8) M), greater than twofold elevations in micronucleus formation were noted in MCF-7 cell cultures that went on to exhibit greater than 75% clonogenic survival; these levels of chromosomal damage were comparable to those induced by 10(-6) M benzo[a]pyrene, a known genotoxic agent. At this low concentration range, a fenitrothion-induced twofold elevation in B-cell leukemia/lymphoma-2 (BCL-2) and cytochrome P450 isoenzyme (CYP1A1) gene expressions was observed. Principal component analysis-linear discriminant analysis (PCA-LDA) of derived infrared (IR) spectra of vehicle control (nonexposed) and OPP-exposed cells highlighted that both fenitrothion and diazinon induced marked biochemical alterations in the lipid, protein, and DNA/RNA absorbance regions. Our findings demonstrate that the two OPP parent chemicals and IMP degradate can mediate a number of toxic effects or cellular alterations at very low concentrations. These are independent of just selective inhibition of AChE, with potential consequences for nontarget organisms exposed at environmentally relevant concentrations. Further assays on relevant aquatic organism cell lines are now recommended to understand the mechanistic low-dose toxicity of these chemicals present in aquatic systems.

Distinguishing cell types or populations based on the computational analysis of their infrared spectra.

Infrared (IR) spectroscopy of intact cells results in a fingerprint of their biochemistry in the form of an IR spectrum; this has given rise to the new field of biospectroscopy. This protocol describes sample preparation (a tissue section or cytology specimen), the application of IR spectroscopy tools, and computational analysis. Experimental considerations include optimization of specimen preparation, objective acquisition of a sufficient number of spectra, linking of the derived spectra with tissue architecture or cell type, and computational analysis. The preparation of multiple specimens (up to 50) takes 8 h; the interrogation of a tissue section can take up to 6 h (∼100 spectra); and cytology analysis (n = 50, 10 spectra per specimen) takes 14 h. IR spectroscopy generates complex data sets and analyses are best when initially based on a multivariate approach (principal component analysis with or without linear discriminant analysis). This results in the identification of class clustering as well as class-specific chemical entities.

Syrian hamster embryo (SHE) assay (pH 6.7) coupled with infrared spectroscopy and chemometrics towards toxicological assessment.

The Syrian hamster embryo (SHE) assay (pH 6.7) is an in vitro candidate to replace in vivo carcinogenicity tests. However, the conventional method of visual scoring of foci (non-transformed vs. transformed colonies) can be time-consuming and is open to subjectivity. Infrared (IR) spectroscopy has the potential to provide objective assessment of such SHE colonies with the added advantage of potentially providing mechanistic information. In this study, SHE cells were treated with one of eight different chemical regimens, allowed in culture to attach and form foci on IR-reflective glass slides; these were subsequently interrogated by attenuated total reflection (ATR) Fourier-transform IR (FTIR) spectroscopy. Derived mid-IR spectra (n = 13,406) were subjected to chemometric analysis focusing primarily on the extraction of biochemical information related to test agent treatment and/or morphological transformation. The use of ATR-FTIR spectroscopy with chemometrics to analyze the SHE assay is a novel approach to toxicological assessment.

Binary mixture effects by PBDE congeners (47, 153, 183, or 209) and PCB congeners (126 or 153) in MCF-7 cells: biochemical alterations assessed by IR spectroscopy and multivariate analysis.

Target organisms are continuously and variously exposed to contaminant mixtures in the environment. We noted that treatment with brominated diphenyl ether (BDE)47 or polychlorinated biphenyl (PCB)126 (toxic equivalency factor [TEF] = 0.1) induces similar alterations in MCF-7 cells when these were determined using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy with multivariate analysis. Because this method appears sensitive enough to signature low-dose effects, we examined how various test agents interact in binary mixtures to induce cell alterations. MCF-7 cells were exposed for 24 h to low concentrations (10(-12) M) of polybrominated diphenyl ether (PBDE) congeners (47, 153, 183, or 209) with or without the coplanar PCB126 or nonplanar PCB153. Following treatment, ethanol-fixed cellular material was interrogated using ATR-FTIR spectroscopy; derived IR spectra in the biochemical-cell fingerprint region (1800 cm(-1)-900 cm(-1)) were then subjected to principal component analysis-linear discriminant analysis. Assuming that if two test agents independently induce the same cell alteration that in combination they'll give rise to an additive effect, we examined predicted versus observed differences in induced alterations by binary mixtures. Compared to corresponding control clusters, treatment with PBDE congener plus PCB126 appeared to cancel out their respective induced alterations. However, treatment with binary mixtures including PCB153 gave rise to an enhanced segregation. Our findings suggest that test agents which mediate their cellular effects via similar mechanisms might result in inhibition within a binary mixture whereas independently acting agents could exacerbate induced alterations in overall cell status.

Polybrominated diphenyl ether-associated alterations in cell biochemistry as determined by attenuated total reflection Fourier-transform infrared spectroscopy: a comparison with DNA-reactive and/or endocrine-disrupting agents.

Whether polybrominated diphenyl ethers (PBDEs) induce effects in target cells is increasingly important given that their environmental burdens are rising. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can be used to biochemically signature cells based on the notion that a detailed "biochemical-cell fingerprint" in the form of an infrared (IR) spectrum is derived. By employing subsequent computational approaches such as principal component analysis (PCA) and/or linear discriminant analysis (LDA), data reduction is achieved to allow for the identification of wavenumber-related biomarkers of effect Clustering of similar spectra (or scores) away from dissimilar ones highlights the variance responsible for discriminating classes. Discriminating biomarkers might include protein conformational changes, structural alterations to DNA/RNA, glycogen content, or protein phosphorylation. Employing this approach, we investigated in MCF-7 cells the dose-related effects of PBDEs (congeners 47, 153, 183, and 209), benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP), 17beta-Oestradiol (E2), or lindane (gamma-hexachlorocyclohexane). Cultures concentrated in G0/G1- or S-phases were treated for 24 h. Following treatment MCF-7 cells were fixed and applied to IR reflective Low-E windows for interrogation using ATR-FTIR spectroscopy. At concentrations as low as 10(-12) M in culture, significant separation (P < or = 0.05) of PBDE-treated and vehicle control cell populations was noted. In some cases this was associated with alterations in lipid or the secondary structure of proteins; with DNA-reactive compounds (e.g., B[a]P), variance was primarily noted in the DNA/RNA region. This study points to a novel nondestructive approach capable of identifying contaminant effects at environmental concentrations in target cells.