The effect of low doses of chlorpyrifos on blood and bone marrow cells in Wistar rats

The study was approved by the Institutional Animal Care and Use Committee and the Croatian Ministry of Agriculture (Reg. No.: 525-10/0255-14-2; file class UP/I-322-01/14-01/75 of 12 September 2014) as well as the Ethics Committee of the Institute for Medical Research and Occupational Health (IMROH) (Reg. No.: 100-21/14-6; file class: 01-18/14-02-2/6 of 11 June 2014). It was carried out in compliance with international standards and national legislation for animal welfare protection.

Chlorpyrifos 99.9 % (CAS No. 2921-88-2) was purchased as analytical standard PESTANAL^® (Sigma-Aldrich Laborchemikalien GmbH; Seelze, Germany). The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA): ehylenediaminetetraacetic acid (EDTA) disodium salt dehydrate, ethyl methane sulphonate (EMS), Tris-HCl, Triton X-100, ethidium bromide, low-melting point (LMP) and normal melting point (NMP) agarose, and acridine orange. Kemika (Zagreb, Croatia) supplied ethanol, NaOH, KCl, and NaCl. Foetal bovine serum (FBS) was purchased from Gibco^® Life Technologies (Grand Island, NY, USA).

Thirty male Wistar rats used in this study were bred until adult age (three months) at the IMROH Animal Breeding Unit (Zagreb, Croatia). We selected only male rats to avoid potential sex-related differences in results. Before the experiment, they were randomised into six groups of five rats. The decision to minimise the sample was based on the EU Directive 2010/63 (55). Body weights of the selected rats were in the range 286–338 g. Rats were kept at 22 °C in stable microenvironmental conditions, with 12 h light/dark cycle. They had free access to tap water and food (4RF21 Complete feed for mice and rats, Mucedola srl, Settimo Milanese, Italy).

The stock pesticide solution was prepared by reconstitution of CPF powder in ethanol and diluted with saline for treatment of rats. The percentage of ethanol in CPF solutions administered to animals did not exceed 0.03 % [this concentration was used as solvent control (SC)].

We tested the effects of CPF at daily doses of 0.010 mg/kg bw (10× the current ADI and AOEL values), 0.015 mg/kg bw (3× the current value of ARfD), and 0.160 mg/kg bw [taken from the World Health Organization (WHO) (56) and the European Food Safety Authority (EFSA) report (57)].

Chlorpyrifos was administered orally with the gastric tube (1 mL of CPF solution per rat a day) for 28 days. Body weights were monitored once a week, and CPF doses adjusted accordingly.

Appropriate negative (NC), SC, and positive control (PC) groups were studied in parallel. Negative control (NC) received 1 mL of PBS instead of CPF. The PC group was receiving a well-known genotoxic agent ethyl methane sulphonate (EMS) (58) at a daily dose of 300 mg/ kg bw over the final three days of the experiment.

All animals were inspected daily by a licensed veterinarian at IMROH to assess survival, clinical signs of toxicity, and body weight changes. Body weight of each animal (expressed in grams) was measured at the beginning and the end of the experiment. Body weight gain was calculated according to the following formula: [(Final weight - Initial weight) × 100] / Initial weight.

The experiment was terminated 24 h after the final gavage. Animals were humanely euthanised by exsanguination under intraperitoneal anaesthesia with a combination of xylazine (Xylapan, 12 mg/ kg bw) and ketamine (Narketan, 80 mg/kg bw), both produced by Vetoquinol UK Ltd. (Towcester, UK). During dissection, the rats were examined by a licensed veterinarian for possible internal organ abnormalities. Blood samples for the in vivo MN assay were collected from the carotid artery, without anticoagulant, and immediately pipetted onto slides pre-coated with acridine orange (AO). Bone marrow samples were collected simultaneously and processed as described below.

AO-coated microscope slides were prepared using a water solution of the stain (1 mg/mL, V =10 μL per slide), which was carefully applied on the surface of the slide, as recommended by Hayashi et al. (59). A drop of blood (V=5 μL) was then pipetted on the pre-coated slide, covered with a coverslip, and stored in a box protecting it from light until the analysis. Slides were analysed for the presence of micronuclei in reticulocytes and erythrocytes with a 1000× magnification fluorescence microscope (Leitz Orthoplan, Oberkochen, Germany) equipped with a blue excitation filter of 502–525 nm.

Reticulocytes, i.e. young RNA-containing erythrocytes (or polychromatic erythrocytes, PCE), emitted orange-red fluorescence. Cells with a few red fluorescent dots were not regarded as reticulocytes. Mature erythrocytes (or normochromatic erythrocytes, NCE) had green colour. Round structures which emitted a bright yellow-green fluorescence were recorded as micronuclei (MN).

Microscopic analysis included MN counts per 2000 polychromatic erythrocytes (PCE), reticulocyte (or PCE) count per 1000 NCE, and MN frequency in 4000 randomly selected reticulocytes per rat (two independent evaluations on two replicate preparations were made).

Both femurs were taken out by dissection, cleaned to remove muscles and other tissues. The epiphyses were cut off and the bone marrow gently flushed with 2 mL of foetal bovine serum (FBS) and aspirated with a syringe, and then centrifuged at 390 g for 10 min). The obtained pellet was washed with phosphate buffered saline (Ca²⁺ and Mg²⁺ free PBS) to remove FBS. Cells were resuspended in PBS and used to prepare slides for the alkaline comet assay.

Microscope slides (Vitrognost Plus Ultra, Biognost, Zagreb, Croatia) pre-coated with 1 % NMP agarose were prepared earlier and stored in a tightly closed plastic box until use. Shortly after collection, bone marrow samples were further processed according to the standard comet assay protocol (60) with some adjustments as reported elsewhere (61). For each experimental group, two replicate preparations were made. The first layer of microgel consisted of 0.6 % NMP agarose. To prepare the second layer, bone marrow cell suspension (V=20 μL per slide) was mixed with 0.5 % LMP agarose (V=100 μL per slide). The top microgel layer consisted of 0.5 % LMP agarose. Preparations were transferred into a cold freshly prepared lysis buffer (2.5 mol/ L NaCl, 100 mmol/ L EDTA, 10 mmol/L Tris, 10 % DMSO, 1 % Triton X-100, pH 10, 4 °C). Lysis lasted for 1 h at +4 °C, and was followed by denaturation in cold alkaline buffer (pH>13), composed of 300 mmol/ L NaOH and 1 mmol/L EDTA. After 20 min of denaturation, the preparations were placed in an electrophoresis unit (Horizon 11.14, Whatman, Florham Park, NJ, USA) filled with the same buffer. The electrophoresis was run for 20 min at +4 °C, and 0.86 V/ cm (61). Thereafter the microgels were neutralised in 0.4 mol/ L Tris-HCl (pH 7.5), rinsed with re-distilled water, dehydrated in 70 % and 96 % ethanol for 10 min each, and allowed to dry at room temperature.

The slides were stained with 20 μg/mL ethidium bromide immediately before analysis under a fluorescence microscope (Olympus BX-51, Olympus, Tokyo, Japan) with a charge coupled device (CCD) camera. Comets were captured at 200× magnification.

Slides were analysed with the Comet Assay IV™ image analysis system (Instem-Perceptive Instruments Ltd., Suffolk, Halstead, UK). One hundred and fifty randomly selected comets per slide were scored (representing a total of 300 comets per animal or 1500 comets per experimental group). Microgel areas near to slide edges or around entrapped air bubbles were excluded from the scoring (62). Since the nucleoids with no or small head and long diffuse tails may indicate cytotoxicity-related DNA damage (63, 64), we recorded only those nucleoids which had <80 % DNA in the tail region. We used tail intensity (% DNA in comet tail) as a descriptor of the DNA damage.

For statistical analysis we used the STATISTICA software (Data Science Workbench, version 14; License No. 14.0.0.15; TIBCO Software Inc., 2020; Palo Alto, CA, USA).

The homogeneity of variances of in vivo MN assay data was tested with Levene’s test at a significance level of 5 %. If variances of the data were homogenous, we used one-way ANOVA. The means were further compared using post hoc Tukey’s HSD test. If variances were not homogenous even after log-normalisation, we used the non-parametric Mann-Whitney U test.

Comet assay results were evaluated with one-way ANOVA, after we tested the normality of distribution with the Shapiro-Wilk test. For post hoc analysis, we used Tukey’s HSD test.

Differences between groups were considered significant when p<0.05.