A study protocol for the evaluation of occupational mutagenic/carcinogenic risks in subjects exposed to antineoplastic drugs: a multicentric project

The wipe sampling procedure will be performed according to the method of Sessink et al. [64], modified [65]. The technique involves cleaning a standard surface measuring 15 × 15 cm (225 cm²) thoroughly using a set of folded wipes (non-woven disposable material) wetted with 2 ml of 0.03 M NaOH solution. The standard sampling sites will be located on the hood surface (preparation site) or the drip surface (administration site). Blank wipes will also be tested. New gloves will be used for each collected wipe sample to prevent cross-contamination. The wipes will be put in test-tubes and transferred to the laboratory. Subsequently, 18 ml of 0.03 M NaOH solution will be added. The samples will be stirred for 10 minutes, sonicated for 30 minutes, centrifuged at 3000 rpm for 20 minutes and stored at -20°C until analysis.

The analysis will be carried out with GC-MS/MS. Briefly, a 1 ml of sample will be added with 100 μl of ifosfamide (IS) as internal standard. The solution will be put in a cartridge containing diatomaceous heart and the analytes eluted with 5 ml of ethyl acetate (twice). Organic layers will be combined and dried in a vacuum centrifuge. The residue will be dissolved in 100 μl of ethyl acetate and the CP and IS will be derivatized by adding 50 μl of trichloroacetic anhydride at 70°C for 30 minutes. The solvent will be evaporated in a vacuum centrifuge, and the residue will be dissolved in 50 μl of toluene and analysed in GC-MS/MS.

The pad technique - widely used for monitoring xenobiotics [5] - will be used to evaluate contamination of clothes of nurses exposed to antineoplastic drugs. A pad of non-woven disposable material will be applied to clothes of the left forearm (non-dominant arm) of each subject during the working shift. The pad will be dampened with 1 ml of 0.03 M NaOH solution. The pads will be recovered at the end of the shift, placed in test-tubes and transferred to the laboratory, where they will be treated according to the procedure used for the wipe-tests.

On the whole, 1 wipe test (hood surface) and 1 pad test (left forearm) will be performed for each worker preparing antineoplastic drugs; 1 wipe test (drip surface) and 1 pad test (left forearm) will be carried out for each worker administering the drugs.

T he determination of urinary 8-hydroxy-2-deoxyguanosine (8OHdG) will be determined in both study groups, i.e. workers exposed and not exposed to antineoplastic drugs. The method, validated by Sabatini et al. [67], is based on high-performance capillary liquid chromatography coupled with an electrospray source with tandem mass spectrometric detection (micro-HPLC-ESI-MS/MS). Urine samples will be collected and extracted using Isolute Env+ SPE cartridges, and 0.5 μl of this extract will undergo micro-HPLC-ESI-MS/MS. MS/MS analysis will be conducted in positive ion mode and acquisition will be performed in MRM mode, selectively monitoring the typical transition of 8OHdG.

To assess primary DNA damage under alkaline conditions, the single-cell microgel electrophoresis (comet) assay [68, 69] will be performed on peripheral blood leukocytes of exposed and control nurses [60, 70‐72]. For slide preparation, 10 μl aliquots of heparinized blood will be mixed with 100 μl low-melting-point agarose (0.7% in PBS) and layered onto pre-treated conventional slides. After overnight lysis (+4°C) in alkaline buffer (pH 10) of cellular and nuclear membranes, the slides will be placed in a horizontal electrophoresis box, allowed to unwind for 20 min in an electrophoretic alkaline buffer (pH > 13) and then subjected to electrophoresis (+4°C) for 20 min by applying an electric field of 1 V/cm and adjusting the current to 300 mA. Lastly, the microgels will be neutralized and stained with ethidium bromide. To evaluate DNA damage, the slides will be examined using an epi-fluorescence microscope equipped with a high-sensitivity CCD (charge-coupled device) camera connected to a computerized image analysis system. Computerized imaging will be performed on coded slides using dedicated software which estimates damage parameters (i.e. tail length, tail intensity and tail moment) by comet profile [73]. Two hundred cells will be analysed for each subject (100 cells/slide, 2 slides per subject). Since the three research units examining DNA damage will use different computerized analysis systems, intercalibration tests will be performed to standardize the procedures used for DNA migration evaluation. For this purpose, the extent of DNA migration will also be evaluated by "visual scoring" based on visual classification of DNA damage.

DNA oxidative damage will be evaluated in peripheral blood lymphocytes with a modification of the standard comet assay using enzymes of the excision repair system [74]. Endonuclease III (Endo-III) recognizes and cuts oxidized bases, mostly pyrimidines [75, 76]. When this enzyme nicks DNA at sites of oxidatively damaged nucleotides, it creates single-strand breaks(SSB) detectable using the alkaline comet assay. For slide preparation, freshly collected white blood cells will be included in agarose microgels as described for the standard comet assay. After the lysis step, the microgels will be incubated with Endo-III for 60 min at 37°C. The slides will undergo alkaline electrophoresis (30 min unwinding and 20 min electrophoresis) and then stained as described for the standard comet assay. To evaluate DNA damage, the slides will be examined as described above for the standard procedure of the comet assay.

A modified protocol that uses a DNA repair inhibitor has been proposed as a means for increasing the sensitivity of the assay [77]. In particular, lymphocyte incubation with cytosine arabinoside (Ara-C) inhibits DNA re-synthesis during nucleotide excision repair and under standard experimental conditions transforms 'cryptic' lesions into SSB detectable with the alkaline comet assay. The evaluation of DNA damage to cells treated with Ara-C will be performed on peripheral blood lymphocytes isolated from whole blood samples using polysucrose density-gradient. The lymphocytes will be re-suspended in RPMI-1640 medium and cultured for 16 h in the presence or the absence of Ara-C (1 μg/ml) [78]. At the end of the culture time the cells will be washed and harvested by centrifugation, and the pellets will be mixed with low-melting-point agarose, subjected to alkaline lysis and electrophoresis, and stained and analysed as described for the standard comet assay.

All the subjects (exposed and controls) will be tested for the presence of MN in lymphocytes. Cell cultures will be set up by adding 0.3 ml of whole blood to 4.7 ml of RPMI-1640 medium supplemented with 20% foetal calf serum, 2 mM L-glutamine, 2% phytohaemoagglutinin and penicillin-streptomycin (100 IU/ml e 100 μg/ml, respectively). Whole blood cultures will be incubated for 72 h at 37°C, 5% CO₂ [79]. To have binucleated cells, cytochalasin B (final concentration 3 μg/ml) will be added after 44 h [80]. The cells will then be collected by centrifugation, re-suspended in a pre-warmed hypotonic solution (75 mM KCl) for 15 min at 37°C and fixed in acetic acid - methanol (1:5 v:v). Air-dried preparations will be stained with 4% Giemsa. For cytogenetic analysis, a total of 1000 binucleated lymphocytes with preserved cytoplasm will be scored for each subject. MN evaluation will be based on standard criteria [81].

The entire population of enrolled subjects will also be tested for the presence of CA. Cell cultures will be set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI-1640 medium supplemented with 10% foetal calf serum, 2% phytohaemoagglutinin and 1.5% penicillin-streptomycin. Whole blood will be cultured at 37°C, 5% CO2, following 90 minutes treatment with 0.2 μg/ml colcemid. 5-Bromodeoxyuridine will be added at a final concentration of 10 μg/ml. Cultures will be fixed at 48 h according to standard protocol [82]. Air-dried metaphase spreads will be stained according to the conventional unbanded Giemsa method. For cytogenetic analysis, an average of 100 well-spread metaphases per subject will be examined by optical microscopy. The analysis of CA will be performed only on cells with 46 chromosomes (+/- 1). CA will classified on the basis of standard criteria [83]. Both chromosome- and chromatid-type aberrations will be scored. Chromosome and chromatid breaks will be distinguished from gaps according to their break size and morphology.

DNA from peripheral blood leucocyte (PBL) pellets will be isolated with a Promega Wizard genomic DNA purification kit (Promega, Italy). As described previously [84], the procedure will provide DNA free of RNA and protein contamination. A multiplex PCR method will be used to detect the presence or absence of the GSTM1 and GSTT1 genes, according to the protocol described previously [85]. This PCR method presents both GSTM1- and GSTT1-specific primer pairs in the same amplification mixture and includes a third primer pair for β-globin as an internal positive PCR control. The GSTT1 (480 bp), β-globin (285 bp), and GSTM1 (215 bp) amplification products will be resolved in an ethidium bromide-stained 2% agarose gel. The absence of the GSTM1- or GSTT1-specific fragment indicates the corresponding null genotype (*0/*0), whereas the β-globin-specific fragment confirms the presence of amplifiable DNA in the reaction mixture. Quality control measures will be adopted for validation of results using RT-PCR and blind repeat of 10% of samples.