Study sensitivity: Evaluating the ability to detect effects in systematic reviews of chemical exposures.

A critical step in systematic reviews of potential health hazards is the structured evaluation of the strengths and weaknesses of the included studies; risk of bias is a term often used to represent this process, specifically with respect to the evaluation of systematic errors that can lead to inaccurate (biased) results (i.e. focusing on internal validity). Systematic review methods developed in the clinical medicine arena have been adapted for use in evaluating environmental health hazards; this expansion raises questions about the scope of risk of bias tools and the extent to which they capture the elements that can affect the interpretation of results from environmental and occupational epidemiology studies and in vivo animal toxicology studies, (the studies typically available for assessment of risk of chemicals). One such element, described here as "sensitivity", is a measure of the ability of a study to detect a true effect or hazard. This concept is similar to the concept of the sensitivity of an assay; an insensitive study may fail to show a difference that truly exists, leading to a false conclusion of no effect. Factors relating to study sensitivity should be evaluated in a systematic manner with the same rigor as the evaluation of other elements within a risk of bias framework. We discuss the importance of this component for the interpretation of individual studies, examine approaches proposed or in use to address it, and describe how it relates to other evaluation components. The evaluation domains contained within a risk of bias tool can include, or can be modified to include, some features relating to study sensitivity; the explicit inclusion of these sensitivity criteria with the same rigor and at the same stage of study evaluation as other bias-related criteria can improve the evaluation process. In some cases, these and other features may be better addressed through a separate sensitivity domain. The combined evaluation of risk of bias and sensitivity can be used to identify the most informative studies, to evaluate the confidence of the findings from individual studies and to identify those study elements that may help to explain heterogeneity across the body of literature.

O-hydroxylamine-coupled alkaline gel electrophoresis assay for the detection and measurement of DNA single-strand breaks.

The ability to detect and measure DNA single-strand breaks has been the aim of numerous assays developed to assess genotoxicity. These methods often rely on alkaline conditions to denature DNA. However, alkaline treatment of DNA also introduces artifactual SSBs through the cleavage of alkali-labile sites resulting in confounded data. Here, we describe a modified alkaline gel electrophoresis assay coupled with a neutral O-hydroxylamine to obtain the measurement of true SSB formation.

Accumulation of true single strand breaks and AP sites in base excision repair deficient cells.

Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase ß null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1mM MMS compared to control. Furthermore, the Pol ß-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays.

Analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine by ultra high pressure liquid chromatography-heat assisted electrospray ionization-tandem mass spectrometry.

Increased amounts of reactive oxygen species (ROS), generally termed oxidative stress, are frequently hypothesized to be causally associated with many diseases. Analyses of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA and urine are widely used biomarkers for oxidative stress. Over the years it became clear that analysis of 8-oxo-dG in DNA is challenging due to artifactual formation during sample work up. The present study demonstrates that 8-oxo-dG can be measured reliably and accurately when appropriate precautions are taken. First, the presence of an antioxidant, metal chelator, or free radical trapping agent during sample preparation improves reproducibility. Second, sample enrichment by HPLC fraction collection was used to optimize sensitivity. Third, heat assisted electrospray ionization (HESI) eliminated potential interferences and improved assay performance and sensitivity. Subsequently, the UPLC-HESI-MS/MS method was applied to show the biphasic dose response of 8-oxo-dG in H(2)O(2)-treated HeLa cells. Application of this method to human lymphocyte DNA (n=156) gave a mean+/-SD endogenous amount of 1.57+/-0.88 adducts per 10(6) dG, a value that is in agreement with the suggested amount previously estimated by European Standard Committee on Oxidative DNA Damage (ESCODD) and others. These results suggest that the present method is well suited for application to molecular toxicology and epidemiology studies investigating the role of oxidative stress.

Cells deficient in the FANC/BRCA pathway are hypersensitive to plasma levels of formaldehyde.

Formaldehyde is an aliphatic monoaldehyde and is a highly reactive environmental human carcinogen. Whereas humans are continuously exposed to exogenous formaldehyde, this reactive aldehyde is a naturally occurring biological compound that is present in human plasma at concentrations ranging from 13 to 97 micromol/L. It has been well documented that DNA-protein crosslinks (DPC) likely play an important role with regard to the genotoxicity and carcinogenicity of formaldehyde. However, little is known about which DNA damage response pathways are essential for cells to counteract formaldehyde. In the present study, we first assessed the DNA damage response to plasma levels of formaldehyde using chicken DT40 cells with targeted mutations in various DNA repair genes. Here, we show that the hypersensitivity to formaldehyde is detected in DT40 mutants deficient in the BRCA/FANC pathway, homologous recombination, or translesion DNA synthesis. In addition, FANCD2-deficient DT40 cells are hypersensitive to acetaldehyde, but not to acrolein, crotonaldehyde, glyoxal, and methylglyoxal. Human cells deficient in FANCC and FANCG are also hypersensitive to plasma levels of formaldehyde. These results indicate that the BRCA/FANC pathway is essential to counteract DPCs caused by aliphatic monoaldehydes. Based on the results obtained in the present study, we are currently proposing that endogenous formaldehyde might have an effect on highly proliferating cells, such as bone marrow cells, as well as an etiology of cancer in Fanconi anemia patients.

Chromium(III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese hamster ovary cells.

Chromium trispicolinate (CrPic) is a popular dietary supplement that is not regulated by the Food and Drug Administration. We are using this compound as a bio-available model to explore the role of Cr(III) in Cr(VI)-induced cancers. The ability of CrPic to cause mutations at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus of CHO AA8 cells has been measured after a 48 h exposure. The highest dose tested was 80 microg/cm(2) CrPic, which, if fully soluble, would be equivalent to 1mM or 0.44 mg/ml CrPic, and would correspond to 1mM Cr(III) or 52 microg/ml Cr(III). This exposure resulted in 68+/-16% cell survival based on 48 h cell counts, and 24+/-11% survival by 7-day colony formation. Exposure of CHO cells to CrPic produced a statistically significant increase in 6-thioguanine (6-TG)-resistant cells over the dose range tested. The 80 microg/cm(2) CrPic exposure resulted in an average induced mutation frequency (MF) of 58 per 10(6) surviving cells, or an average 40-fold increase in hprt mutants relative to untreated cells. An equivalent dose of 3mM Pic was highly cytotoxic and did not yield hprt mutants. The dose range of 0.375-1.5mM Pic produced a slight increase in hprt mutants, but the increase was not statistically significant. An equivalent dose of 1mM chromic chloride yielded an induced MF of 9 per 10(6) surviving cells, or a 10-fold increase in mutants with cell survivals of >100%. The coordination of Cr(III) with picolinic acid may make the metal more genotoxic than other forms of Cr(III). In light of the current results and the known ability of Cr(III) and CrPic to accumulate in tissues, as well as the growing evidence of Cr(III) involvement in Cr(VI)-induced cancers, we caution against ingestion of large doses of CrPic for extended periods.