High-throughput in vivo genotoxicity testing: an automated readout system for the somatic mutation and recombination test (SMART).

Genotoxicity testing is an important component of toxicity assessment. As illustrated by the European registration, evaluation, authorization, and restriction of chemicals (REACH) directive, it concerns all the chemicals used in industry. The commonly used in vivo mammalian tests appear to be ill adapted to tackle the large compound sets involved, due to throughput, cost, and ethical issues. The somatic mutation and recombination test (SMART) represents a more scalable alternative, since it uses Drosophila, which develops faster and requires less infrastructure. Despite these advantages, the manual scoring of the hairs on Drosophila wings required for the SMART limits its usage. To overcome this limitation, we have developed an automated SMART readout. It consists of automated imaging, followed by an image analysis pipeline that measures individual wing genotoxicity scores. Finally, we have developed a wing score-based dose-dependency approach that can provide genotoxicity profiles. We have validated our method using 6 compounds, obtaining profiles almost identical to those obtained from manual measures, even for low-genotoxicity compounds such as urethane. The automated SMART, with its faster and more reliable readout, fulfills the need for a high-throughput in vivo test. The flexible imaging strategy we describe and the analysis tools we provide should facilitate the optimization and dissemination of our methods

Automated SMART process.

<p>The methods used to automate the SMART are depicted with a flow diagram. The general flow of the method is described in (A). Each step in the process is depicted by a rectangle, with data inputs and outputs depicted using elliptical shapes. (B) Steps in automated acquisition. (C) Steps in focus stack analysis and the extraction of data on <i>mwh</i> hairs, cells, and spots. Discard error (1–3) refers to detection of (1) abnormal hair shape, (2) abnormal hair orientation, and/or (3) abnormal position relative to the wing surface. (D) Steps in the construction of the genotoxicity wing score dose-response curve and the characterization of compound genotoxicity.</p

<i>mwh</i> hair detection.

<p>All segmented hairs are characterized by vectors prior to the detection of <i>mwh</i> phenotypes. (A) A schematic side view of the wing illustrates hair organization, with hairs on each side of the wing surface (dashed gray line): top (green) and bottom (blue). A close-up of this schematic (A, orange circle) illustrates that (B) locally, the wing can be considered flat. Its altitude (z₀) can be estimated from local hair roots. Hair root altitude relative to the surface (z_{s =} z-z₀) and angle relative to the surface (<i>φ</i>)are used to determine the wing side. Indeed, both the <i>φ</i> distribution (D) and the z_s distribution (E, green curve) present two modes corresponding to top and bottom hairs. Absolute hair root altitude (z) distribution (E, red curve) does not show such a separation. (C) presents a bird’s eye view of hair organization on the top side of the wing. Detection of <i>mwh</i> hairs relies on their small inter-hair distance (red circle and arrow). The distribution of relative positions of adjacent hairs illustrates this characteristic. (F–G) show distributions built from control and total samples, respectively. (H–I) show details of these distributions. These distributions adhere to the color map in (K). Only their left or right side is shown, due to distribution symmetry, and their highest density is set to one in each image, allowing more details to be seen in (H–I). Distributions may look similar, but their close-ups are distinct; the distribution determined in total samples has a specific mode that corresponds to the <i>mwh</i> hair phenotype. A distance threshold (green circle) allows us to select <i>mwh</i> hairs using that mode. (J) An example of hair wing side detection (bottom hairs in blue, top hairs in green) is overlaid on a focus stack detail. (L) A side view of the section of (J) is presented in the red, dashed rectangle. (M) An example of <i>mwh</i> hair detection (red arrows) is overlaid on a manually constructed image of wing bottom hairs.</p

Genotoxicity analysis.

<p>DRCs for 6 test compounds. Manual measures (blue crosses) and automated measures (green triangles) were obtained, allowing us to detect genotoxic (A, B, and C) and non-genotoxic (D, E, and F) compounds. Known genotoxins (first row) all exhibited increasing genotoxic effects with increasing concentration. Further analyses with fitting a sigmoidal curve to the data (A, B, and C; solid lines) and 95% confidence envelopes (dashed lines) allowed the EC₅₀ and slope parameters to be determined, facilitating characterization of compound genotoxicity. Findings from manual and automated measurements were highly correlated after scaling. Non-genotoxic compounds (second row) showed no significant dose-dependence (linear fit shown as a solid line) in either manual or automated measures.</p

SMART automated image and data acquisition.

<p>To test chemical-induced genotoxicity, fly larvae (A) were exposed to increasing doses (B; c0, ci, for instance) of a test compound and their wings (C), along with those of other flies from their treatment group, were collected on a slide. A single wing is shown in the red rectangle. (D) Wing position and orientation (orange arrow) were detected automatically, and acquisition regions were defined (green rectangles, each corresponding to a microscope field of view). All acquisition positions defined were compiled in a single file used by the microscope to perform multipoint acquisitions. (E) At each point, a focus stack showing wing hairs and their spatial organization was acquired. (F) A close-up of a focus stack maxima projection along the focus axis, showing hair organization. Hairs from the upper and lower sides of the wing overlap in this view. (G) For illustrative purposes, lower wing hairs have been separated by manually selecting a set of z-slices before projection. Focusing on a single wing side, one can distinguish the regular position and orientation of the hairs. Hairs with an <i>mwh</i> phenotype are visible (white dashed circle).</p